Surfaces and coating compositions having antifouling, antithrombotic, and antibacterial properties and methods of making

ABSTRACT

Coating compositions, coated articles including the coating compositions, and methods of making the coating compositions and coated articles are provided. In some aspects, the coating compositions are applied to a substrate having nitric oxide-releasing properties. The coating compositions can include copolymers having crosslinking agents that can be activated with mild UV light (about 345 nm to 365 nm) to avoid damaging the substrate while creating strong covalent bonds to the substrate. The copolymers can include hydrophilic repeat units, and in particular zwitterionic repeat units such as repeat units containing phosphorylcholine groups. In some aspects, the coating compositions are applied to a surface of a polymer substrate, wherein the polymer substrate had nitric oxide releasing properties. The coating compositions and the coated articles can have antifouling, antithrombotic, and/or antibacterial properties.

CROSS-REFERENCE TO RELATED APPLICATIONS

This application is the 35 U.S.C. § 371 national stage of PCTapplication having serial number PCT/US2018/056778, filed on Oct. 19,2018; which application also claims priority to, and the benefit of,U.S. provisional application entitled “SURFACES HAVING ANTIFOGGING ANDANTIFOULING CHARACTERISTICS, COATING COMPOSITIONS HAVING ANTIFOGGING ANDANTIFOULING CHARACTERISTICS, AND METHODS OF MAKING ANTIFOGGING ANDANTIFOULING SURFACES” having Ser. No. 62/617,418, filed Jan. 15, 2018;U.S. provisional application entitled “SURFACES AND COATING COMPOSITIONSHAVING ANTIFOULING CHARACTERISTICS, AND METHODS OF MAKING” having Ser.No. 62/575,104, filed Oct. 20, 2017; and U.S. provisional applicationentitled “SURFACES AND COATING COMPOSITIONS HAVING ANTIFOULING,ANTITHROMBOTIC, AND ANTIBACTERIAL PROPERTIES AND METHODS OF MAKING”having Ser. No. 62/685,621, filed Jun. 15, 2018, the contents of whichare incorporated by reference in their entireties.

STATEMENT REGARDING FEDERALLY SPONSORED RESEARCH OR DEVELOPMENT

This invention was made with Government support under contractsK25HL111213, R01HL134899, and R01HL111213 awarded by the NationalInstitutes of Health and contract 200-2016-91933 awarded by the Centersfor Disease Control and Prevention. The Government has certain rights inthe invention.

TECHNICAL FIELD

The present disclosure generally relates to coatings, coatingcompositions, coated articles, and methods of use and making thereof.

BACKGROUND

The non-specific adsorption of proteins has long been considered a grandchallenge in many biomedical applications such as implants, contactlenses, catheters, and biosensors. In addition to medical devicefailure, the consequences of protein adsorption include thrombusformation, innate immune response, and bacterial infection. Preventingdirect microbial contamination is also highly desired characteristic ofmedical devices, implants, and hospital equipment. Although significantprogress has been made in understanding and reducing adsorption andcontamination, the Centers for Disease Control and Prevention (CDC)still reported that, in 2011, there were an estimated 722,000healthcare-associated infections (HAIs) in U.S. acute care hospitals.Additionally, about 75,000 patients with HAIs died during theirhospitalization. On any given day, approximately 1 out of every 25patients in the U.S. contracts at least one infection during theirhospital care. Therefore, materials demonstrating antifouling andantimicrobial effects are highly desirable.

There remains a need for improved coating technologies, coatingcompositions, coatings, and coated articles that overcome theaforementioned deficiencies.

SUMMARY

In various aspects described herein, coating compositions, coatedarticles, and methods of making and using thereof are provided. Invarious aspects, the coated articles provide mechanisms for both activeand passive antifouling, antimicrobial, and/or antithromboticproperties.

In some aspects, the coated articles include a nitric oxide-releasingsubstrate having at least one surface; and a zwitterionic polymercovalently attached to the at least one surface to form the coatedarticle. The NO release can provide for prolonged active antifouling,antimicrobial, and/or antithrombotic properties. By presenting thezwitterionic groups on the surface, the coated articles are also capableof providing passive antifouling, antimicrobial, and/or antithromboticproperties that persist even once the NO release from the substrate hasceased.

In some aspects, the zwitterionic polymer is a copolymer comprisingzwitterionic repeat units and tethering repeat units, and the tetheringrepeat units include covalent bonds to the at least one surface of thesubstrate. For instance, the zwitterionic polymer can be a randomcopolymer having a structure according to the following formula:

where each occurrence of Z is a zwitterionic moiety; where, in eachinstance, either (i) A¹ is none and A² is ═O or (ii) A¹ is a covalentbond to the at least one surface of the substrate and A² is —OH; whereeach occurrence of R¹ is independently a covalent bond or a linear orbranched, substituted or unsubstituted alkyl diradical having from 1 to12 carbon atoms; where each occurrence of R², R³, and R⁴ isindependently a linear or branched, substituted or unsubstituted alkylhaving from 1 to 12 carbon atoms; and where a, b, and c are real numbersuch that 0<a<1, 0≤b<1, 0<c<1, and a+b+c=1.

Suitable nitric oxide-releasing substrates can include a polymer andnitric-oxide donor dispersed within the polymer; wherein thenitric-oxide donor is present in an amount from about 6 wt % to about 11wt % based upon a total weight of the nitric oxide-releasing substrate.In some aspects, the substrates have surface hydrogens capable offorming covalent bonds to the tethering repeat units. The nitric-oxidedonor dispersed in the polymer can be any suitable NO donor materialsuch as an organic nitrate, a metal-NO complex, an N-nitrosamine, anS-nitrosothiol, or a combination thereof.

In some aspects, the coated articles include a substrate having nitricoxide-releasing groups covalently attached to the surface throughbranched alkylamine spacers. The alkylamine spacers can be covalentlyattached to the surface of the substrate such that the branchedalkylamine spacer coupled to the surface of the polymer substrateprovides passive antifouling, antithrombotic and antimicrobialproperties, while the NO-donor moiety releases NO and provides activeantimicrobial properties.

In various aspects, coating compositions including the polymers areprovided as well as methods of making coated articles using the coatingcompositions. In particular, the polymers can include crosslinkingagents that can be activated with mild UV light (about 345 nm to 365 nm)to avoid damaging the substrate while creating strong covalent bonds tothe substrate. The methods can include applied the polymers to thesurface using spin coating, spray coating, dip coating, pad application,or films with adhesive backing. The methods can include applying UVlight to crosslink the polymers to the surface of the substrate.

Coated articles can include any article where antifouling,antithrombotic and antimicrobial properties are beneficial. In someaspects, the article can include a catheter (including, but not limitedto, vascular and urinary catheters), a coronary stent, a wound dressing,extracorporeal circuits, membrane oxygenators, endotracheal tubes, avascular graft, and the like.

Other systems, methods, features, and advantages of the coatingcompositions, coated articles, and methods of making thereof will be orbecome apparent to one with skill in the art upon examination of thefollowing drawings and detailed description. It is intended that allsuch additional systems, methods, features, and advantages be includedwithin this description, be within the scope of the present disclosure,and be protected by the accompanying claims.

BRIEF DESCRIPTION OF THE DRAWINGS

Further aspects of the present disclosure will be readily appreciatedupon review of the detailed description of its various embodiments,described below, when taken in conjunction with the accompanyingdrawings.

FIG. 1 shows nitric oxide chemiluminescence analyzer flowchart.

FIG. 2 shows UV-vis absorption spectrum of BPMPC drop-cast onto a quartzsubstrate as a function of photochemical irradiation time at 254 nm (6.5mW cm⁻² intensity).

FIG. 3 shows ATR-FTIR spectra of BPMPC coatings before (lower curve) andafter (upper curve) UV exposure.

FIG. 4 shows contact angle measurement as a function of time forCarboSil coated with BPMPC and incubated at 37° C. in PBS under mildagitation.

FIGS. 5A-5B show SNAP leaching measured using UV-vis over 2 weeks andNitric oxide release measured over 2 weeks (n=3) usingchemiluminescence.

FIGS. 6A-6B show thickness increase after incubation in (FIG. 6A)Fibrinogen solution and in (FIG. 6B) Lysozyme solution.

FIGS. 7A-7B show fluorescence micrographs (magnification 10×) ofuncoated films after (FIG. 7A) 90-minute incubation, (FIG. 7B) 1 day inBSA PBS solution before incubation, and (FIG. 7C) 7 days in BSA PBSsolution before incubation in 2 mg/ml FITC-BSA solution. (FIGS. 7D-7F)are the coated film measured under the same experimental conditions.

FIG. 8 shows antimicrobial efficacy of NO-releasing BPMPC coated samplesrelative to controls (n=3).

FIG. 9 shows the fabrication of the SNAP-BPAM CarboSil film and itsbiocidal action. Antibacterial polymeric composites were fabricated byincorporating a NO donor, S-nitroso-N-acetyl-penicillamine (SNAP) inCarboSil polymer and top coated with surface immobilized benzophenonebased quaternary ammonium (BPAM) antimicrobial small molecule viaphotocrosslinking.

FIGS. 10A-10B show UV-mediated photocrosslinking study of BPAM on SNAPfilm: (FIG. 10A): UV-Vis Spectra of SNAP-BPAM film before and after UVirradiation (254 nm, 90 s). After UV irradiation for 90 seconds,absorbance at 255 nm decreased, indicating the completion of thecrosslinking reaction. (FIG. 10B): Total SNAP content after UVirradiation was reported to be approximately 95.44±2.5% of the initialSNAP content. The data is reported as a mean±standard deviation for n=3samples and the significance with a p-value <0.05 is stated forcomparisons.

FIG. 11 shows real-time NO flux rate of SNAP doped CarboSil (black) andBPAM coated SNAP CarboSil films (red) analyzed at the physiologicaltemperature using Sievers Nitric Oxide Analyzer (NOA).

FIG. 12 shows nitric oxide (NO) flux analysis of SNAP and SNAP-BPAMfilms (at 0 h and 24 h time-period). The SNAP-BPAM films released higherNO flux at initially and at 24 h time point. The NO flux of SNAP-BPAMwas maintained in the physiological range even after 24 h. The data isreported as a mean±standard deviation for n=3 samples and thesignificance with a p-value <0.05 is stated for comparisons.

FIG. 13 shows comparative graphs to show the differences in inhibitionof viable colony forming units of Gram-positive S. aureus andGram-negative P. aeruginosa on the unit surface area (CFU/cm²) of SNAPfilms, BPAM films and SNAP-BPAM films as compared to control CarboSil.The results suggest that both SNAP and BPAM has different degree oftoxicity towards gram positive and negative bacteria. While BPAM byitself has better antibacterial potential towards gram negative P.aureginosa as compared to SNAP, SNAP is better than BPAM w.r.t itsbactericidal action against gram positive S. aureus. The combined actionof SNAP-BPAM works equally well against both the bacteria. The data isreported as a mean±standard deviation for n=3 samples and thesignificance with a p-value <0.05 is stated for comparisons.

FIG. 14 shows the effect of immobilized BPAM top coats on NO releasekinetics of the SNAP-BPAM films as measured by chemiluminescence before(0 h and 24 h) and after bacterial exposure (24 h) study. The residualNO flux was observed to be in the physiological range even after 24 h ofbacteria exposure indicating the antibacterial effect can be extendedbeyond the 24 h. The data is reported as a mean±standard deviation forn=3 samples and the significance with a p-value <0.05 is stated forcomparisons.

FIGS. 15A-15B show the zone of inhibition (ZOI) can be seen inside thedotted red circle: (FIG. 15A) S. aureus; (FIG. 15B) P. aeruginosa; (i)Control, (ii) SNAP, (iii) BPAM, and (iv) SNAP-BPAM. The bigger ZOI withSNAP-BPAM combination is due to increase in NO flux with BPAM topcoat.

FIG. 16 shows preparation of surface immobilizedS-nitroso-N-acetyl-d-penicillamine (SIM-S1, SIM-S2, SIM-S4) A)Functionalization of PDMS surface with hydroxyl groups by submerging itin 50:50 ratio of 13 N HCl: 30 wt. % H2O2 in H2O B) Treatment withAPTMES for amine functionalization C) Ring-opening reaction ofNAP-thiolactone with free amine groups to produce free thiol groups(repeated for step G and K for SIM-N2 and SIM-N4, respectively) D)Nitrosation of thiol groups with tert-butyl nitrite (repeated for step Hand L for SIM-S2 and SIM-S4, respectively) E) Branching of primary aminevia reaction with methyl acrylate (repeated for step I) F) Aminefunctionalization of branched site using ethylene diamine (repeated forstep J).

FIG. 17 shows FTIR spectra for different samples. Amine-1, Amine-2 andAmine-4 correspond to amine functionalized surfaces with unbranched andbranched surfaces. 3500-2500 represents unreacted —COOH groups presentafter amine-functionalization for SIM-N4 and Amine-2. 2950 representsalkyl groups present in abundance in SR and aminated surfaces of SR.Double peaks of 1650, 1550 represent primary amine groups in Amine-1,Amine-2 and Amine-4. 1650 represents saturated amide groups in SIM-N1,SIM-N2, SIM-N4, SIM-S1, SIM-S2 and SIM-S4. 1550 in SIM-S2 representsnitroso group of the NO-donor attached.

FIG. 18 shows an illustration of embodiments of un-branched (SIM-S1) andbranched (SIM-S2 and SIM-S4) surface immobilized NO-donor surfaces ofthe present disclosure.

FIGS. 19A-19B show (FIG. 19A) day by day NO release measurements for 600h/25 d and (FIG. 19B) Cumulative NO release over 600 h/25 d.

FIG. 20 shows adsorption of fibrinogen to modified SR surfaces over a 2h period. Values are expressed as mean±standard error. Measurements wereconducted using n=8 per group. *—significantly different compared tounmodified SR. #—significantly different compared to SIMS-2configuration.

FIG. 21 shows that SIM-S2 was able to reduce bacteria adhesion by ˜2.5logs when compared to control samples.

FIG. 22 show platelets adsorbed per surface area over a period of 90mins.

DETAILED DESCRIPTION

In various aspects, coating compositions, coated articles, and methodsof making thereof are provided that overcome one or more of theaforementioned problems. In accordance with the purpose(s) of thepresent disclosure, as embodied and broadly described herein,embodiments of the present disclosure relate to coatings, substrates,and/or articles having antifouling and antimicrobial characteristics,methods of making the coatings, substrate, and/or articles, and methodsof decreasing the amount of biochemical components and/or microorganismsformed on a surface of the substrate or article, and the like.

Before the present disclosure is described in greater detail, it is tobe understood that this disclosure is not limited to particularembodiments described, and as such may, of course, vary. It is also tobe understood that the terminology used herein is for the purpose ofdescribing particular embodiments only, and is not intended to belimiting. The skilled artisan will recognize many variants andadaptations of the embodiments described herein. These variants andadaptations are intended to be included in the teachings of thisdisclosure and to be encompassed by the claims herein.

All publications and patents cited in this specification are cited todisclose and describe the methods and/or materials in connection withwhich the publications are cited. All such publications and patents areherein incorporated by references as if each individual publication orpatent were specifically and individually indicated to be incorporatedby reference. Such incorporation by reference is expressly limited tothe methods and/or materials described in the cited publications andpatents and does not extend to any lexicographical definitions from thecited publications and patents. Any lexicographical definition in thepublications and patents cited that is not also expressly repeated inthe instant specification should not be treated as such and should notbe read as defining any terms appearing in the accompanying claims. Thecitation of any publication is for its disclosure prior to the filingdate and should not be construed as an admission that the presentdisclosure is not entitled to antedate such publication by virtue ofprior disclosure. Further, the dates of publication provided could bedifferent from the actual publication dates that may need to beindependently confirmed.

Although any methods and materials similar or equivalent to thosedescribed herein can also be used in the practice or testing of thepresent disclosure, the preferred methods and materials are nowdescribed. Functions or constructions well-known in the art may not bedescribed in detail for brevity and/or clarity. Embodiments of thepresent disclosure will employ, unless otherwise indicated, techniquesof nanotechnology, organic chemistry, material science and engineeringand the like, which are within the skill of the art. Such techniques areexplained fully in the literature.

It should be noted that ratios, concentrations, amounts, and othernumerical data can be expressed herein in a range format. It is to beunderstood that such a range format is used for convenience and brevity,and thus, should be interpreted in a flexible manner to include not onlythe numerical values explicitly recited as the limits of the range, butalso to include all the individual numerical values or sub-rangesencompassed within that range as if each numerical value and sub-rangeis explicitly recited. To illustrate, a numerical range of “about 0.1%to about 5%” should be interpreted to include not only the explicitlyrecited values of about 0.1% to about 5%, but also include individualvalues (e.g., 1%, 2%, 3%, and 4%) and the sub-ranges (e.g., 0.5%, 1.1%,2.2%, 3.3%, and 4.4%) within the indicated range. Where the stated rangeincludes one or both of the limits, ranges excluding either or both ofthose included limits are also included in the disclosure, e.g. thephrase “x to y” includes the range from ‘x’ to ‘y’ as well as the rangegreater than ‘x’ and less than ‘y’. The range can also be expressed asan upper limit, e.g. ‘about x, y, z, or less’ and should be interpretedto include the specific ranges of ‘about x’, ‘about y’, and ‘about z’ aswell as the ranges of ‘less than x’, less than y′, and ‘less than z’.Likewise, the phrase ‘about x, y, z, or greater’ should be interpretedto include the specific ranges of ‘about x’, ‘about y’, and ‘about z’ aswell as the ranges of ‘greater than x’, greater than y′, and ‘greaterthan z’. In some embodiments, the term “about” can include traditionalrounding according to significant figures of the numerical value. Inaddition, the phrase “about ‘x’ to ‘y’”, where ‘x’ and ‘y’ are numericalvalues, includes “about ‘x’ to about ‘y’”.

Definitions

Abbreviations: BPMPC, 2-methacryloyloxyethyl phosphorylcholine-co-butylmethacrylate-co-benzophenone;N-(4-benzoylbenzyl)-N,N-dimethylbutan-1-ammonium iodide (BPAM); NO,nitric oxide; SNAP, S-nitroso-N-acetylpenicillamine; BP,4-vinylbenzophenone; MPC, 2-Methacryloyloxyethyl phosphorylcholine; BSA,bovine serum albumin; FITC-BSA, fluorescein isothiocyanate labeledbovine serum albumin; NAP, N-acetyl-D-penicillamine; NaNO₂, sodiumnitrite; conc. H₂SO₄, concentrated sulfuric acid; THF, tetrahydrofuran;NaH₂PO₄, sodium phosphate monobasic; LB, Luria broth; Na₂HPO₄, sodiumphosphate dibasic; EDTA, ethylenediamine tetraacetic acid; NaOH, sodiumhydroxide; KH₂PO₄, potassium phosphate monobasic; CarboSil® 20 80Athermoplastic silicone-polycarbonate-urethane (hereafter will bereferred to as CarboSil); DMAc, dimethylacetamide; NBS,N-Bromosuccinimide; AlBN, 2, 2′-azo-bis(2-methylpropionitrile); PBS,Phosphate buffered saline; ATCC, American Type Tissue CultureCollection.

The terms “anti-fouling” or “anti-foul” as used herein, applies tocompositions, surfaces, or articles having characteristics preventing orminimizing the adhesion of biological materials (e.g., proteins),microorganisms, or other debris.

The terms “antimicrobial” and “antimicrobial characteristic” refers tothe ability to kill and/or inhibit the growth of microorganisms. Asubstance having an antimicrobial characteristic may be harmful tomicroorganisms (e.g., bacteria, fungi, protozoans, algae, and the like).A substance having an antimicrobial characteristic can kill themicroorganism and/or prevent or substantially prevent the growth orreproduction of the microorganism.

The terms “bacteria” or “bacterium” include, but are not limited to,Gram positive and Gram negative bacteria. Bacteria can include, but arenot limited to, Abiotrophia, Achromobacter, Acidaminococcus, Acidovorax,Acinetobacter, Actinobacillus, Actinobaculum, Actinomadura, Actinomyces,Aerococcus, Aeromonas, Afipia, Agrobacterium, Alcaligenes, Alloiococcus,Alteromonas, Amycolata, Amycolatopsis, Anaerobospirillum, Anabaenaaffinis and other cyanobacteria (including the Anabaena, Anabaenopsis,Aphanizomenon, Camesiphon, Cylindrospermopsis, Gloeobacter Hapalosiphon,Lyngbya, Microcystis, Nodularia, Nostoc, Phormidium, Planktothrix,Pseudoanabaena, Schizothrix, Spirulina, Trichodesmium, and Umezakiagenera) Anaerorhabdus, Arachnia, Arcanobacterium, Arcobacter,Arthrobacter, Atopobium, Aureobacterium, Bacteroides, Balneatrix,Bartonella, Bergeyella, Bifidobacterium, Bilophila, Branhamella,Borrelia, Bordetella, Brachyspira, Brevibacillus, Brevibacterium,Brevundimonas, Brucella, Burkholderia, Buttiauxella, Butyrivibrio,Calymmatobacterium, Campylobacter, Capnocytophaga, Cardiobacterium,Catonella, Cedecea, Cellulomonas, Centipeda, Chlamydia, Chlamydophila,Chromobacterium, Chyseobacterium, Chryseomonas, Citrobacter,Clostridium, Collinsella, Comamonas, Corynebacterium, Coxiella,Cryptobacterium, Delftia, Dermabacter, Dermatophilus, Desulfomonas,Desulfovibrio, Dialister, Dichelobacter, Dolosicoccus, Dolosigranulum,Edwardsiella, Eggerthella, Ehrlichia, Eikenella, Empedobacter,Enterobacter, Enterococcus, Erwinia, Erysipelothrix, Escherichia,Eubacterium, Ewingella, Exiguobacterium, Facklamia, Filifactor,Flavimonas, Flavobacterium, Francisella, Fusobacterium, Gardnerella,Gemella, Globicatella, Gordona, Haemophilus, Hafnia, Helicobacter,Helococcus, Holdemania, Ignavigranum, Johnsonella, Kingella, Klebsiella,Kocuria, Koserella, Kurthia, Kytococcus, Lactobacillus, Lactococcus,Lautropia, Leclercia, Legionella, Leminorella, Leptospira, Leptotrichia,Leuconostoc, Listeria, Listonella, Megasphaera, Methylobacterium,Microbacterium, Micrococcus, Mitsuokella, Mobiluncus, Moellerella,Moraxella, Morganella, Mycobacterium, Mycoplasma, Myroides, Neisseria,Nocardia, Nocardiopsis, Ochrobactrum, Oeskovia, Oligella, Orientia,Paenibacillus, Pantoea, Parachlamydia, Pasteurella, Pediococcus,Peptococcus, Peptostreptococcus, Photobacterium, Photorhabdus,Phytoplasma, Plesiomonas, Porphyrimonas, Prevotella, Propionibacterium,Proteus, Providencia, Pseudomonas, Pseudonocardia, Pseudoramibacter,Psychrobacter, Rahnella, Ralstonia, Rhodococcus, Rickettsia RochalimaeaRoseomonas, Rothia, Ruminococcus, Salmonella, Selenomonas, Serpulina,Serratia, Shewenella, Shigella, Simkania, Slackia, Sphingobacterium,Sphingomonas, Spirillum, Spiroplasma, Staphylococcus, Stenotrophomonas,Stomatococcus, Streptobacillus, Streptococcus, Streptomyces,Succinivibrio, Sutterella, Suttonella, Tatumella, Tissierella,Trabulsiella, Treponema, Tropheryma, Tsakamurella, Turicella,Ureaplasma, Vagococcus, Veillonella, Vibrio, Weeksella, Wolinella,Xanthomonas, Xenorhabdus, Yersinia, and Yokenella. Other examples ofbacterium include Mycobacterium tuberculosis, M. bovis, M. typhimurium,M. bovis strain BCG, BCG substrains, M. avium, M. intracellulare, M.africanum, M. kansasii, M. marinum, M. ulcerans, M. avium subspeciesparatuberculosis, Staphylococcus aureus, Staphylococcus epidermidis,Staphylococcus equi, Streptococcus pyogenes, Streptococcus agalactiae,Listeria monocytogenes, Listeria ivanovii, Bacillus anthracis, B.subtilis, Nocardia asteroides, and other Nocardia species, Streptococcusviridans group, Peptococcus species, Peptostreptococcus species,Actinomyces israelii and other Actinomyces species, andPropionibacterium acnes, Clostridium tetani, Clostridium botulinum,other Clostridium species, Pseudomonas aeruginosa, other Pseudomonasspecies, Campylobacter species, Vibrio cholera, Ehrlichia species,Actinobacillus pleuropneumoniae, Pasteurella haemolytica, Pasteurellamultocida, other Pasteurella species, Legionella pneumophila, otherLegionella species, Salmonella typhi, other Salmonella species, Shigellaspecies Brucella abortus, other Brucella species, Chlamydi trachomatis,Chlamydia psittaci, Coxiella burnetii, Escherichia coli, Neisseriameningitidis, Neisseria gonorrhea, Haemophilus influenzae, Haemophilusducreyi, other Hemophilus species, Yersinia pestis, Yersiniaenterocolitica, other Yersinia species, Escherichia coli, E. hirae andother Escherichia species, as well as other Enterobacteria, Brucellaabortus and other Brucella species, Burkholderia cepacia, Burkholderiapseudomallei, Francisella tularensis, Bacteroides fragilis,Fusobacterium nucleatum, Provetella species, and Cowdria ruminantium, orany strain or variant thereof. The Gram-positive bacteria may include,but is not limited to, Gram positive Cocci (e.g., Streptococcus,Staphylococcus, and Enterococcus). The Gram-negative bacteria mayinclude, but is not limited to, Gram negative rods (e.g.,Bacteroidaceae, Enterobacteriaceae, Vibrionaceae, Pasteurellae andPseudomonadaceae). In an embodiment, the bacteria can include Mycoplasmapneumoniae.

The term “protozoan” as used herein includes, without limitationsflagellates (e.g., Giardia lamblia), amoeboids (e.g., Entamoebahistolytica), and sporozoans (e.g., Plasmodium knowlesi) as well asciliates (e.g., B. coli). Protozoan can include, but it is not limitedto, Entamoeba coli, Entamoeba histolytica, lodamoeba buetschlii,Chilomastix mesnili, Trichomonas vaginalis, Pentatrichomonas homini,Plasmodium vivax, Leishmania braziliensis, Trypanosoma cruzi,Trypanosoma brucei, and Myxosporidia.

The term “algae” as used herein includes, without limitations microalgaeand filamentous algae such as Anacystis nidulans, Scenedesmus sp.,Chlamydomonas sp., Chlorella sp., Dunaliella sp., Euglena sp.,Prymnesium sp., Porphyridium sp., Synechococcus sp., Botryococcusbraunii, Crypthecodinium cohnii, Cylindrotheca sp., Microcystis sp.,Isochrysis sp., Monallanthus salina, M. minutum, Nannochloris sp.,Nannochloropsis sp., Neochloris oleoabundans, Nitzschia sp.,Phaeodactylum tricornutum, Schizochytrium sp., Scenedesmus obliquus, andTetraselmis sueica as well as algae belonging to any of Spirogyra,Cladophora, Vaucheria, Pithophora and Enteromorpha genera.

The term “fungi” as used herein includes, without limitations, aplurality of organisms such as molds, mildews and rusts and includespecies in the Penicillium, Aspergillus, Acremonium, Cladosporium,Fusarium, Mucor, Neurospora, Rhizopus, Trichophyton, Botryotinia,Phytophthora, Ophiostoma, Magnaporthe, Stachybotrys and Uredinalesgenera.

The terms “broad-spectrum biocide”, “biocide”, and “biocidal” as usedherein include, without limitation, pesticides (e.g. fungicides,herbicides, insecticides, algicides, molluscicides, miticides, androdenticides) and antimicrobials and may also include germicides,antibiotics, antibacterials, antivirals, antifungals, antiprotozoals,and antiparasites.

The term “antimicrobial effective amount” as used herein refers to thatamount of the compound being administered/released which will killmicroorganisms or inhibit growth and/or reproduction thereof to someextent (e.g. from about 5% to about 100%). In reference to thecompositions or articles of the disclosure, an antimicrobial effectiveamount refers to that amount which has the effect of diminishment of thepresence of existing microorganisms, stabilization (e.g., notincreasing) of the number of microorganisms present, preventing thepresence of additional microorganisms, delaying or slowing of thereproduction of microorganisms, and combinations thereof. Similarly, theterm “antibacterial effective amount” refers to that amount of acompound being administered/released that will kill bacterial organismsor inhibit growth and/or reproduction thereof to some extent (e.g., fromabout 5% to about 100%). In reference to the compositions or articles ofthe disclosure, an antibacterial effective amount refers to that amountwhich has the effect of diminishment of the presence of existingbacteria, stabilization (e.g., not increasing) of the number of bacteriapresent, preventing the presence of additional bacteria, delaying orslowing of the reproduction of bacteria, and combinations thereof.

As used herein the term “biocompatible” refers to the ability toco-exist with a living biological substance and/or biological system(e.g., a cell, cellular components, living tissue, organ, etc.) withoutexerting undue stress, toxicity, or adverse effects on the biologicalsubstance or system.

NO, nitric oxide; SNAP, S-nitroso-N-acetylpenicillamine; GSNO,S-nitroso-glutathione; PVA, polyvinyl alcohol; SIM, Surface immobilized

Unless defined otherwise, all technical and scientific terms used hereinhave the same meaning as commonly understood by one of ordinary skill inthe art to which this disclosure belongs. It will be further understoodthat terms, such as those defined in commonly used dictionaries, shouldbe interpreted as having a meaning that is consistent with their meaningin the context of the specification and relevant art and should not beinterpreted in an idealized or overly formal sense unless expresslydefined herein.

The articles “a” and “an,” as used herein, mean one or more when appliedto any feature in embodiments of the present invention described in thespecification and claims. The use of “a” and “an” does not limit themeaning to a single feature unless such a limit is specifically stated.The article “the” preceding singular or plural nouns or noun phrasesdenotes a particular specified feature or particular specified featuresand may have a singular or plural connotation depending upon the contextin which it is used.

Coating Compositions

The present disclosure includes a coating comprising a NO-donorsubstrate and a polymer. In an aspect, the polymer includes ahydrophilic moiety and a photo cross-linkable moiety, where the coatinghas one or both of anti-fouling and antimicrobial characteristics. In anaspect, the hydrophilic moiety is covalently attached to the surface ofthe NO-donor substrate through a photo cross-linkable moiety uponexposure to light energy (e.g., ultraviolet energy), which can form acoating (e.g., about 10 to 500 nm thick) on the surface. Advantageously,the NO-donor substrate has antimicrobial properties, and the photocross-linkable moiety has antifouling characteristics. The attachment ofthe photo cross-linkable moiety to the NO-donor substrate is a one stepprocess which is simple and scalable.

In an embodiment, the photo cross-linkable moiety can include an arylketone (about 340 to 400 nm), an aryl azide group (about 250 to 450 nmor about 350 to 375 nm), a diazirine group (about 340 to 375 nm), andthe polymer can include a combination of these groups. In an embodiment,the photo cross-linkable moiety can include alkyl-arylketones anddiarylketones bearing at least one condensed ring system substituentsuch as naphthyl and anthracenyl. In an embodiment, the aryl ketonegroup can include benzophenone (about 340 to 380 nm), acetophenone(about 340 to 400 nm), a naphthylmethylketone (about 320 to 380 nm), adinaphthylketone (about 310 to 380 nm), a dinaphtylketone derivative(about 320 to 420 nm), or derivatives of each of these. In anembodiment, the photo cross-linkable moiety is a benzophenone group. Inan embodiment, the aryl azide group can include phenyl azide, alkylsubstituted phenyl azide, halogen substituted phenyl azide, orderivatives of each of these. In an embodiment, the diazirine group caninclude 3,3 dialkyl diazirine (e.g., 3,3 dimethyl diazirine, 3, 3diethyl diazirine), 3,3 diaryl diazirine (e.g., 3,3 diphenyl diazirine),3-alkyl 3-aryl diazirine, (e.g., 3-methyl-3-phenyl diazirine), orderivatives of each of these.

Embodiments of the present disclosure include a coating as above, wherethe photo cross-linkable moiety can be benzophenone. Hydrophilicpolymers can be used as coating materials for the preparation ofsuperhydrophilic surfaces. The ability of an aryl ketone moiety such asbenzophenone (BP) to act as a cross-linking agent and abstract hydrogenfrom a suitable hydrogen donor has been well studied and utilized invarious chemical systems for many years.5-7 BP can be used forcrosslinking organic thin films and it can be activated using mild UVlight (345-365 nm), avoiding oxidative damage of the polymer and surfacethat can occur upon exposure to higher energy UV. The benzophenonemoiety is more chemically robust than other organic cross-linkers andreacts preferentially with C—H bonds in a wide range of differentchemical environments. Triggered by UV light, benzophenone undergoes ann-pi* transition, resulting in the formation of a biradical tripletexcited state that can abstract a hydrogen atom from a neighboringaliphatic C—H group to form a new C—C bond. This triplet state also hasespecially high reactivity for H located alpha to electron donatingheteroatoms (nitrogen and oxygen). This photoreaction has recently beenused to attach thin polymer layers to metal and oxide surfaces,8-11along with applications in microfluidics,12 organic semiconductors,13and biosensors.14 (the references for this paragraph correspond toExample 1)

In an embodiment, the hydrophilic moiety functions to provide at least ahydrophilic characteristic to the coating. Embodiments of the presentdisclosure include a surface as above, where the hydrophilic moiety canbe a C1-C4-alkyl methacrylate such as iso-butyl methacrylate or alkylacrylate.

In an embodiment the hydrophilic moiety can be a zwitterionic polymer(or ethylene glycol polymer, or hydroxyfunctional acrylates polymer). Invarious embodiments, the zwitterionic polymer can be2-methacryloyloxyethyl phosphorylcholine-co-butylmethacrylate-co-benzophenone (BPMPC) (or any sulfobetaine methacrylate,carboxybetaine methacrylate-co-butyl methacrylate-co-benzophenonecopolymers). The BPMPC can be from about 20% to about 99%2-methacryloyloxyethyl phosphorylcholine (MPC), (e.g. 30% MPC, 50% MPC,70% MPC, or 90% MPC). Advantageously, the BPMPC coating has excellenthydrophilicity, which helps inhibit the adsorption of proteins fromsolution when the coating is applied to an article. In variousembodiments, the BPMPC coating is from about 20 nm to about 1 μm.

In an embodiment the polymer can be represented by one or more of thefollowing polymers:

where the ratio of the components can vary from those described above,where each subscript can vary from 0.05 to 0.9.

Substrates

In an aspect, the NO-donor substrate releases NO. Embodiments of thepresent disclosure include a coating as above, where the NO-donorsubstrate includes an organic nitrate, a metal-NO complex, anN-nitrosamine, an S-nitrosothiol, or a combination thereof. In variousembodiments, NO-donor substrate can be a polymer film doped withS-nitroso-N-acetylpenicillamine (SNAP). Embodiments of the presentdisclosure include a coating as above, where the NO-donor substrate hasabout 6%-11% wt S-nitroso-N-acetylpenicillamine (SNAP). In variousembodiments, the polymer film can be silicone-polycarbonate-urethanethermoplastic (CarboSil).

Advantageously, the NO-donor substrate releases NO at a higher, moresustained rate when coated with the BPMPC than when used alone. Anotheradvantage to the coating is the reduced leaching of SNAP over CarboSilalone.

Embodiments of the present disclosure include a coating as above,wherein the NO-donor substrate is covalently linked to the photocross-linkable moiety upon exposure to light energy. In an embodiment,the photo cross-linkable moiety and the NO-donor substrate of coatingare covalently bonded via the interaction of a UV light (e.g., about 340to 370 nm) that causes a C—C bond to form between the NO-donor substrateand the photo cross-linkable moiety.

Embodiments of the present disclosure include a coating as above,wherein the doped polymer film is coated in additional layers of polymerfilm. In various embodiments, the doped polymer film can be coated withone or more layers of film. The coating can be from about 20 nm to about1 μm thick.

The present disclosure also includes substrate having a surface,including a coating comprising an NO-donor substrate and a photocross-linkable moiety. The NO-donor substrate is covalently attached tothe photo cross-linkable moiety to form a film applied to the surface.Advantageously, the coating gives the surface one or both of ananti-fouling and an antimicrobial characteristic.

Although NO-donor substrates provide an active mechanism ofantimicrobial action, once the NO-releasing properties of asubstrate/coating/material have been exhausted, it may no longer provideantifouling/anti-microbial activity. Thus, the present disclosureprovides polymer substrate surfaces, coatings, and articles having bothactive and passive antifouling, antimicrobial, and antithromboticproperties. The present disclosure provides polymer materials and/orcoatings that provide a branched polymer surface chemistry to providehydrophobic and steric hindrances to adhesion by proteins, bacteria andother microbes, while simultaneously providing active antifouling andantimicrobial activity from surface immobilized NO-donors.

In embodiments, the present disclosure provides a polymer materialand/or substrate having a surface-immobilized (SIM) NO-donorfunctionalized surface. The SIM NO-donor functionalized surface includesa branched alkylamine spacer coupled to the surface of the polymersubstrate to provide passive antifouling, antithrombotic andantimicrobial properties. The SIM NO-donor functionalized surface alsoincludes an NO-donor moiety coupled to the branched alkyl spacer suchthat the NO-donor moiety releases NO and provides active antimicrobialproperties. In embodiments, the polymer material having the SIM NO-donorfunctionalized surface retains antifouling, antithrombotic andantimicrobial properties after all of the NO has been released.

In embodiments polymer substrate/polymer material can include polymerssuch as, but not limited to polyurethane, silicone, polyvinyl chloride,ketone polymers (including, but not limited to, polyether ether ketone),polyethylene (including, but not limited to, ethylene vinyl acetate),bioresorbable polymers (including, but not limited to, polylactic acid,polyglycolic acid, and polycaprolactone), fluoropolymers (including, butnot limited to, polytetrafluoroethylene, perfluoroether, and fluorinatedethylene propylene), and combinations thereof. In embodiments, thepolymer substrate includes polymer materials typically used in themaking of medical devices. In embodiments, the polymer substrate is orforms part of a medical device.

In embodiments, the branched alkylamine spacers have multiple branches.The branching steps, described below, can add 1-4 branches to thespacers. Thus, multiple branches are possible, and embodiments of thepresent disclosure are intended to encompass various branchingdensities.

In embodiments, the NO-donor is biocompatible. In embodiments theNO-donor includes biocompatible NO-donors such as, but not limited to,N-nitrosoamine, S-nitrosothiol, diazeniumdiolate, or a combinationthereof. In embodiments a combination of NO-donors can be used. Forinstance, in embodiments, nitrosothiols and diazeniumdiolates can beused in conjunction by having secondary amines in the backbone, withattachment of a nitrosothiol to the end functional group. Inembodiments, the NO-donor is S-nitroso-N-acetylpenicillamine (SNAP),S-nitroso-glutathione, S-nitroso-N-acetylcysteine, among others. In someembodiments the polymer substrate also includes a silicone oilimpregnated into the polymer substrate. In embodiments, the silicon oilis impregnated into the polymer substrate after formation of the SIMNO-donor functionalized surface.

Embodiments of the present disclosure also include medical devicesincluding the polymer substrate of the present disclosure. Such medicaldevices include implantable devices or other medical equipment. In someembodiments, the implantable medical device can include, but is notlimited to a catheter (including, but not limited to, vascular andurinary catheters) a coronary stent, a wound dressing, extracorporealcircuits, membrane oxygenators, endotracheal tubes, a vascular graft,and the like.

The present disclosure also includes articles having a surface with acoating composition, where the coating composition includes a branchedalkylamine spacer coupled to the surface of the polymer substrate and anSIM NO-donor moiety coupled to the alkyl spacer to form an SIM branchedNO-donor coating. With such coatings, the NO-donor moiety releases NO,providing the surface with antifouling, antithrombotic, andantimicrobial properties as well as the properties provided by thebranched polymer spacer described above. In embodiments, the article isa medical implant, medical device, or medical equipment. In embodiments,the article is further impregnated with silicone oil, which increasesthe hydrophobicity of the surface, further increasing the antifouling,anti-thrombotic, and anti-microbial properties. In embodiments, theNO-donor can include, but is not limited to, an N-nitrosoamine, anS-nitrosothiol, a diazeniumdiolate, or a combination thereof. Inembodiments, the NO-donor is S-nitroso-Nacetylpenicillamine (SNAP).

The present disclosure also provides methods of making thecoatings/surfaces of the present disclosure. In embodiments the polymermaterial/substrate/surface can be but is not limited to, polyurethane,silicone, polyvinyl chloride, and combinations thereof. Embodiments ofmethods of making a SIM NO-donor functionalized coating on a polymersubstrate surface include the following general steps. First, thepolymer surface is amine-functionalized. In embodiments,amine-functionalization is preceded by functionalizing the polymersurface with hydroxyl groups to provide a hydroxyl-functionalizedpolymer surface to assist in amine functionalization.

In embodiments the polymer material includes silicone or the polymer hasa silicone coating, or the polymer substrate is a silicone film/coating.In embodiments a silicone polymer surface can be hydroxyl functionalizedby contacting or submerging the silicone surface with a mixture of HCland hydrogen dioxide in water. In embodiments, thehydroxyl-functionalized polymer surface is then functionalized withamine groups to provide an amine-functionalized polymer surface. Inembodiments, amine-functionalization includes contacting thehydroxylfunctionalized surface with APTMES to provide theamine-functionalized surface. Alternatives for imparting hydroxyl groupson the surface include, but are not limited to, treatment with air oroxygen plasma. Application of the silane can be done in solution asmentioned above or through vapor deposition (incubating the sample wherethe silane is vaporized rather than liquid form).

After providing an amine-functionalized polymer surface, in embodiments,the primary amines on the amine-functionalized surface are reacted withan alkyl acrylate to form branched alkyl spacers to provide a branchedsurface. In embodiments, this can include incubating theamine-functionalized surface in methanol: methyl acrylate to formbranched alkyl spacers immobilized to the polymer surface. Afterbranching, the branched alkyl spacers can be functionalized with aminesor alkyl amines to provide an amine functionalized branched polymersurface. In embodiments, this can include incubating the branchedsurface in methanol:ethylenediamine to form amine functionalizedbranched alkyl spacers immobilized to the polymer surface. Inembodiments, the branching and amine functionalization of the branchedspacers is repeated to increase the branching of the spacers. It can berepeated as needed to achieve a desired branching density. Inembodiments, the selection of alkyl acrylate and the selection ofamine/alkyl amine can control the degree of branching. For example, theuse of pentane-1,3,5 triamine rather than ethylenediamine can be used toprovide an additional free primary amine after the methacrylate step.This would then change the branching sequence from 1-2-4, to 1-4-8.Thus, each branching step can add multiple branches and can be repeated(e.g., methacrylate or ethylenediamine) to add more branches, asdesired. In embodiments, each branching step can add 1-4 branches (1branch being a bifunctional molecule).

The NO-donor is then coupled to the immobilized, branched,amine-functionalized alkyl spacers. In embodiments, after providingimmobilized, branched, amine-functionalized, alkyl spacers, the aminefunctionalized branched alkyl spacers are reacted with an NO-donorprecursor to immobilize the NO-donor precursor to the polymer surfacefollowed by nitrosating the NO-donor precursor. In embodiments, theamine functionalized branched polymer surface can be contacted withNAP-thiolactone (a NO-donor precursor), where ring opening ofthiolactone allows binding of the free amines to immobilize NAP to thebranched polymer surface. Nitrosating the NO-donor precursor convertsthe precursor into an active NO-donor and thus provides thesurface-immobilized NO-donor functionalized coating on the polymersurface. In embodiments, the NO-donor precursor is NAP, and nitrosationof the immobilized NAP includes incubating the surface in neattert-Butyl nitrate. The nitrosation step produces a surface-immobilizedSNAP functionalized branched polymer coating on the polymer surface.

Coated Articles and Methods of Making Thereof

The present disclosure includes articles having a surface that has oneor both of antifouling and antimicrobial characteristics. Theantifouling and antimicrobial characteristics are the result of applyinga coating on the surface of the article. As used herein, the one or bothof antifouling and antimicrobial characteristics of the coating preventor substantially reduce (e.g., about 80-99%, about 85 to 99%, about 90to 99%, about 95 to 99.9%) biochemical (e.g., protein) and bacterialaccumulation on a surface relative to the surface without theseantifouling and antimicrobial characteristics. Advantageously, thecoating can be applied to surfaces that can be hydrophilic orhydrophobic.

In an embodiment, the substrate or article (or the coating disposed on asurface of the article) may have an antimicrobial characteristic (e.g.,kills at least 70%, at least 80%, at least 90%, at least 95%, or atleast 99% of the microorganisms (e.g., bacteria) on the surface and/orreduces the amount of microorganisms that form or grow on the surface byat least 70%, at least 80%, at least 90%, at least 95%, or at least 99%,as compared to a similar surface without the coating disposed on thesurface). In an embodiment, the substrate or article (or the coatingdisposed on a surface of the article) may have an anti-foulingcharacteristic (e.g. reduces the amount of biochemical (e.g., proteins),microorganisms, or debris that form or grow on the surface by at least70%, at least 80%, at least 90%, at least 95%, or at least 99%, ascompared to a similar surface without the coating disposed on thesurface).

In various embodiments, the articles can have surfaces including thosethat may be exposed to microorganisms and/or that microorganisms mighttypically grow on such as, without limitation, medical instruments,medical implants, prosthetic devices, contact lenses, plastic devices,circuitry, fabrics, cooking counters, food processing facilities,kitchen utensils, food packaging, packaging materials (e.g., food, meat,poultry, and the like food packaging materials), plastic structures(e.g., made of a polymer or a polymer blend), glass or glass likestructures having a functionalized layer (e.g., includes a C—H group) onthe surface of the structure, metals, metal alloys, or metal oxidesstructure having a functionalized layer (e.g., includes a C—H group) onthe surface of the structure, a structure (e.g., tile, stone, ceramic,marble, granite, or the like) having a functionalized layer (e.g.,includes a C—H group) on the surface of the structure, and a combinationthereof, textiles, filters, marine vessels, swimming pools, metals, drugvials, yarns, fibers, gloves, furniture, toys, diapers, leather, tiles,and flooring materials.

Embodiments of the present disclosure include articles or substrates asabove, where the coating is applied to the surface using e.g. spincoating, spray coating, dip coating, pad application, films withadhesive backing, and the like.

The present disclosure also includes methods of making the substrate byexposing a NO-donor substrate to a polymer including a hydrophilicmoiety and a photo cross-linkable moiety, and exposing the polymer tolight energy, thereby causing the photo cross-linkable moiety tocovalently attach to the NO-donor substrate and form the coating on theNO-donor substrate

The present disclosure also includes methods of making a coating,including doping a polymer film with S-nitroso-N-acetylpenicillamine toform a NO-donor substrate, combining the NO-donor substrate with apolymer to form the coating, and exposing the coating to light energy,thereby causing the photo cross-linkable moiety to covalently attach tothe NO-donor substrate.

Embodiments of the present disclosure include methods as above, whereinthe doped polymer film can be coated in additional layers of polymerfilm. In various embodiments, the doped polymer film can be coated withone or more layers of film. The coating can be from about 20 nm to about1 μm thick.

Nitric oxide (NO) is an endogenous, gaseous, free radical that isproduced naturally by macrophages and by endothelial cells lining thevascular walls, and is involved in various biological processes, such aspreventing platelet activation and adhesion, while also being a potent,broad spectrum bactericidal agent. To take advantage of theseproperties, NO-donors (e.g. s-nitrosothiols or diazeniumdiolates) havebeen developed to allow for the storage and localized delivery of NO,and are particularly advantageous for polymeric materials typically usedfor medical devices, such as polyurethanes, silicones, or polyvinylchloride. The addition of these donors at various levels also provides asimple method for controlling the level of NO that is delivered from thematerials._([15]) Materials releasing NO have been shown tosignificantly reduce thrombus formation in both extracorporeal-circuitand vascular catheter models, and have been shown to provide significantreductions in bacteria during long term catheterization._([2, 16])

EXAMPLES

Now having described the embodiments of the present disclosure, ingeneral, the following Examples describe some additional embodiments ofthe present disclosure. While embodiments of the present disclosure aredescribed in connection with the following examples and thecorresponding text and figures, there is no intent to limit embodimentsof the present disclosure to this description. On the contrary, theintent is to cover all alternatives, modifications, and equivalentsincluded within the spirit and scope of embodiments of the presentdisclosure.

Example 1

To explore the covalent grafting of zwitterionic polymers to varioussubstrates ranging from hydrophilic to hydrophobic, the benzophenone(BP) chromophore, a photoactive tethering reagent, was incorporated intothe polymeric backbone.¹⁹⁻²⁴ The BP group can produce a diradical underlow-intensity UV irradiation (350-365 nm) that abstracts an aliphatichydrogen from a neighboring C—H bond to form a new C—C bond, withoutintensive UV oxidative damage to the polymer or substrates.²⁰ Throughthis process, network polymer films can be grafted with excellentdurability to a broad selection of C—H containing materials andsurfaces, and has been used for many applications such asmicrofluidics,²⁵⁻²⁶ organic semiconductors,²⁷ redox polymers,²⁸⁻²⁹anti-icing polymers,³⁰ and biosensors.³¹⁻³²

In an example of the present disclosure, zwitterionic terpolymers(2-methacryloyloxyethyl phosphorylcholine-co-butylmethacrylate-co-benzophenone, BPMPC) were synthesized that can becovalently grafted to antimicrobial, NO-releasing CarboSil(silicone-polycarbonate-urethane thermoplastic) upon UV-irradiation. Thepolymer-coated surfaces are characterized in detail and the zwitterionicstability is assessed under physiological conditions. The proteinrepellency properties of these coatings are evaluated. At the same time,no SNAP degradation was observed during coating or UV irradiation, andthe release profile remained above the physiological level for 2 weekswith the zwitterionic top-coat. Moreover, enhanced antimicrobialactivity was demonstrated with bacteria testing.

Experiment Section

Materials

4-vinylbenzophenone (BP) was synthesized according to a previouslyreported method.³⁰ 2-Methacryloyloxyethyl phosphorylcholine (MPC),albumin from bovine serum (BSA), fluorescein isothiocyanate labeledbovine serum albumin (FTIC-BSA), N-acetyl-D-penicillamine (NAP), sodiumnitrite (NaNO₂), concentrated sulfuric acid (conc. H₂SO₄),tetrahydrofuran (THF), sodium phosphate monobasic (NaH₂PO₄), sodiumphosphate dibasic (Na₂HPO₄), potassium chloride, sodium chloride, andethylenediamine tetraacetic acid (EDTA) were purchased from SigmaAldrich (St. Louis, Mo.). 2,2′-azobis(2-methylpropionitrile) (AlBN) andn-butyl methacrylate (BMA) were bought from Alfa-Aesar (Haverhill,Mass.). Isobutyltrichlorosilane was purchased from Tokyo ChemicalIndustry (Portland, Oreg.). Concentrated hydrochloric acid (conc. HCl),sodium hydroxide (NaOH), and methanol were bought from Fisher-Scientific(Hampton, N.H.). Potassium phosphate monobasic (KH₂PO₄) and lysozymefrom egg white were purchased from BDH Chemicals-VWR International (WestChester, Pa.). CarboSil™ 20 80A UR STPU (referred to as CarboSil hereon)was acquired from DSM Biomedical Inc. (Berkeley, Calif.). Milli-Q filterwas used to obtain de-ionized (DI) water for all the aqueous solutionpreparations. Nitrogen and oxygen gas cylinders were purchased fromAirgas (Kennesaw, Ga.). Staphylococcus aureus (ATCC 6538, S. aureus) wasused for the bacterial experiments. LB Agar (LA), Miller and Luria broth(LB), Lennox were purchased from Fischer BioReagents (Fair Lawn, N.J.).All the chemicals were used without further purification.

In brief, CarboSil polymers with 10 wt % SNAP (test samples) and no SNAPcontent (control samples) were prepared using solvent evaporation and/orspin coating method. These samples were then coated with a zwitterioniccopolymer (referred to as BPMPC) which was covalently bonded to theCarboSil base polymers by UV-crosslinking. Surface analysis wasperformed on the films pre- and post-UV radiation to understand thecrosslinking behavior of the polyzwitterionic system. Test and controlsamples with the BPMPC coating were analyzed for their NO releasebehavior. The samples were then tested for protein adhesion for 14 daysin physiological conditions (37° C. in PBS) to evaluate antifoulingproperties of the topcoat. Finally, antimicrobial assay of the sampleswas done using a modified version of ASTM E2180 protocol.

Synthesis of NO Donor, SNAP

S-nitroso-N-acetylpenicillamine was synthesized using a revised approachfor a method previously reported.³⁸ 1M H₂SO₄ and 1M HCl were mixed withan equimolar amount of NAP, methanol and NaNO₂ aqueous solution. Thisreaction mixture was stirred for 20 minutes and then cooled for 7 hourswith a constant flow of air on the mixture. After evaporation of theunreacted portion of the reaction mixture, precipitated green crystalsof SNAP were filtered, collected and dried in a covered vacuumdesiccator. Dried crystals of SNAP were used for all experiments.

Synthesis of CarboSil Films Doped with SNAP

CarboSil films containing 10 wt % SNAP were prepared using solventevaporation method. 700 mg of CarboSil was dissolved in 10 mL of THF tomake the polymer solutions. 77 mg of SNAP was added to this solution fora final concentration of 10 wt % of SNAP. This polymer-SNAP blend wasstirred in dark conditions until the SNAP crystals dissolved completely.The blend was then transferred into Teflon molds and allowed to let thesolvent evaporate overnight in fume hood. The overnight dried films werethen cut into circular shapes of 0.8 cm diameter each. Each sample wasimmersed into a CarboSil solution without SNAP (40 mg mL⁻¹ of polymerconcentration in THF) to coat it (this was repeated thrice for eachsample). The samples were dried overnight and then dried under vacuumfor an additional 24 hours. This added drying time was included toeliminate any remaining THF which can affect any following studies.Weight of each film was recorded before the topcoat application for allSNAP leaching behavior tests. The formulated samples were stored in thefreezer (−18° C.) in the dark between experiments to prevent escape ofSNAP or consequent loss of NO. These SNAP-incorporated films were usedfor NO release, SNAP leaching and bacterial cell viability analyses. Allsamples used for the tests were less than a week old to ensure integrityof studies.

Synthesis of Zwitterionic Copolymer (BPMPC)

The polymer was synthesized by free radical polymerization. MPC (0.546g, 1.85 mmol), n-BMA (0.105 mL, 0.66 mmol) and BP (0.027 g, 0.132 mmol)were dissolved in 5.3 mL ethanol (total monomer concentration 1.0 mmolmL⁻¹) with initiator AlBN (0.01 mmol mL⁻¹) and the solution was pouredinto polymerization tube. After degassing with argon for 30 minutes, thepolymerization reaction was carried out under nitrogen flow at 60° C.for 16 h. The reaction was stopped by exposing the solution to air,cooled to room temperature, and poured into ethyl ether to precipitatethe polymer. The white solid was collected by vacuum filtration anddried under vacuum for 12 h. Yield: 0.552 g, 83%. ¹H NMR (D₂O) was takento confirm the polymer composition.

Crosslinking of BPMPC with Substrates

Silicon substrates were cut into 2.4 cm×2.4 cm pieces and sonicated withdeionized water, isopropanol, and acetone for 5 min each then driedunder nitrogen, followed by plasma (Harrick Plasma PDC-32G) clean andtreated with iBTS in toluene overnight before modification with thepolymer. CarboSil substrates were coated with polymer withoutpretreatment.

Two coating methods were utilized when applying BPMPC on substrates:spin coating and spray coating. For spin coating, polymer modified filmwas developed on functionalized silicon substrate by using 0.5 mLBPMPC/ethanol solution (10 mg mL⁻¹) at 1000 rpm for 30 seconds. Spraycoating was applied for CarboSil films with and without SNAP.BPMPC/ethanol solution (2 mg mL⁻¹) was sprayed using a spray gun from adistance of 10 cm onto vertically placed substrates to achieve uniformcoating upon drying. Spin coating was used in the protein adsorptionexperiments, and spray coating in SNAP/NO release and bacterialexperiments, based on method that afforded the smoothest, pin-hole freecoating on different forms of substrate. Then the BPMPC substrates wereirradiated with UV light (UVP, 254 nm, 6.5 mW cm⁻²) for 1 min tocovalently bond the BPMPC to the surface. The substrates were rinsedwith abundant ethanol to remove unattached BPMPC then dried undernitrogen.

Characterization of the Polymer Coatings

The surface wettability was characterized by measuring the static watercontact angle, which obtained from a DSA 100 drop shape analysis system(KRÜSS) with a computer-controlled liquid dispensing system. 1 μL DIwater droplets were deposited onto substrate surfaces, and the watercontact angles were measured within 10 seconds through the analysis ofphotographic images. The cross-linking kinetics of BPMPC coating wasinvestigated by a UV-vis spectroscopy (Varian) with 254 nm UV light. Thethickness of the spin-coated polymer layer on the silicon substrates andCarboSil substrates were measured by M-2000V Spectroscopic Ellipsometer(J.A. Woollam co., INC.) with a white light source at three incidentangles (65°, 70°, and 75°). The thickness of the modified layer wasmeasured and calculated using a Cauchy layer model. Infraredspectroscopy studies of polymer coated films were done using aThermo-Nicolet model 6700 spectrometer equipped with a variable anglegrazing angle attenuated total reflection (GATR-ATR) accessory (HarrickScientific).

SNAP Leaching Study and NO-Release Profile

The percentage of SNAP discharged from the samples were quantified bynoting the absorbance of the PBS solutions (used to soak the samples) at340 nm (characteristic absorbance maxima of S—NO group of SNAP). Eachsample was weighed before coating with non-SNAP polymer solutions todetermine the initial amount of SNAP in each film. The films were thenimmersed in vials containing PBS (pH 7.4 with 100 μM EDTA to preventcatalysis of NO release by metal ions) and stored at 37° C. A UV-visspectrophotometer (Thermoscientific Genesys 10S UV-vis) was utilized toquantify the absorbance of the buffer solutions in the required timeintervals. The readings were converted to wt % of SNAP in the bufferutilizing the initial amount of SNAP present in each sample. 1 mLaliquots of the PBS solution in which the samples were soaked was usedfor each sample absorbance measurement to avoid any inconsistentreadings and three replicates were utilized for each quantification. Thecalibration graph with known amounts of SNAP in PBS (with EDTA) was usedto interpolate the absorbance quantifications recorded from the studyand convert them to concentrations of SNAP in the quantified sample.

SNAP incorporated in the polymers release NO in physiological conditionsand this release was measured and recorded in real time for the studyusing Sievers chemiluminescence NO analyzers® (NOA 280i, GE Analytical,Boulder, Colo., USA). The sample holder maintained dark conditions forthe samples to prevent catalysis of the NO production by any lightsource. It was filled with 5 mL of PBS (pH 7.4 with 100 μM of EDTA) tosoak the samples. EDTA acted as a chelating agent to prevent catalysisof NO production by metal ions in the PBS. This buffer solution wasmaintained at 37° C. by a temperature-regulated water jacket placedaround the sample holder. Once a baseline of NO flux without the sample(prepared according to Example 2: Nitric Oxide release kinetics.) isestablished, the sample is then placed in the sample holder. Nitricoxide released by the sample in the sample holder was pushed and purgedtowards the analyzer by a continuous supply of nitrogen gas maintainedat a constant flow rate of 200 mL min⁻¹ through the sweep and bubbleflows. The NO released by the sample is pushed towards thechemiluminescence detection chamber where the reactions shown on FIG. 1take place.

The voltage signal produced is converted to concentration of NO anddisplayed on the analyzer's screen. Using the raw data in ppb form andNOA constant (mol ppb⁻¹ s⁻¹), the data in ppb is normalized for surfacearea of the sample and converted to NO flux units (×10⁻¹⁰ mol cm⁻²min⁻¹). Data was collected in the time intervals mentioned and sampleswere stored in a PBS (with EDTA) solution at 37° C. in dark conditionsbetween measurements. The PBS was replaced daily to avoid anyaccumulation of SNAP leached or NO released during the storage time. Theinstrument operating parameters were a cell pressure of 7.4 Torr, asupply pressure of 6.1 psig and a temperature of −12° C. Threereplicates were used for each measurement.

Protein Adhesion Assay

Protein adsorption test is a significant important method for evaluatingthe blood adhesion. Therefore, the thickness change of substrates beforeand after incubation in protein solutions was monitored, as anindication of protein adsorption. Coated substrates were incubated infibrinogen (1 mg mL⁻¹) and lysozyme (1 mg mL⁻¹) in PBS (pH 7.4, 0.01 M)solutions up to 14 days, followed by thickness measurement every day.

In the second approach, fluorescein isothiocyanate-bovine serum albumin(FITC-BSA, 2 mg mL⁻¹) in PBS solution was used to evaluate the proteinadsorption behavior on the surface of CarboSil substrate modified byBPMPC.⁴²⁻⁴³ Substrates were immersed in FITC-BSA solution for one andhalf hour at 37° C., then rinsed with distilled water and dried withnitrogen. The substrates with protein then analyzed by Nikon EclipseNI-U fluorescence microscope (Nikon Instruments, Inc.), using a 5×objective lens, with filter set (Ex/Em 470/525 nm). To confirm thelong-term resistance to protein adsorption, the substrates wereincubated in BSA (1 mg ml⁻¹) PBS solution for up to 7 days at 37° C.before putting in FITC-BSA solution.

Bacterial Assay

Bacterial adhesion for each of the samples was calculated in terms ofthe bacterial cell viability using serial dilution after an incubationperiod of 24 hours. The method used to perform this assay was based on amodified version of the American Society for Testing and Materials E2180protocol. S. aureus was used for antimicrobial evaluation of thesamples. Bacteria were cultured in LB Broth (Lennox) at 37° C. and grownto ˜10⁶ colony-forming units (CFU) per mL as measured by opticaldensity. The resulting overnight culture was collected by centrifugation(2500 g, 7 min) and resuspended in PBS. This resuspended bacterialsuspension was used for incubation of polymer samples for 24 hours.

After incubation with the bacterial solution, samples were washed gentlywith PBS to remove any unbound bacteria. The samples were then placed in1 mL of PBS and homogenized for 1 minute each to transfer any adheredbacteria to this new PBS solution. After homogenization, homogenatesamples were serially diluted and plated onto LB Agar nutrient plates(37° C.). Bacterial viability was determined by counting the colonies oneach plate manually. Calculation of bacterial adhesion was done bycounting number of colonies per cm² of each sample.

Statistical Analysis

All data are quantified as mean±standard deviation with an n≥3 for alltrials. The results between the control and test films were analyzed bya comparison of means using student's t-test. Values of p were obtainedfor the data analyzed and p<0.05 was considered significant.

Results and Discussion

The zwitterionic polymer (BPMPC) was synthesized by radicalpolymerization in ethanol (Scheme 1A). The copolymer composition wasconfirmed by ¹H NMR spectroscopy, and consisted of 74:18:8(MPC:nBMA:BP), which roughly matched the monomer feed ratio. This ratioprovided the optimal anti-fouling result (discussed below) along withthe most uniform coating on both hydrophobic and hydrophilic substrates.The polymer synthesis is simple and straightforward, no furtherpurification is required besides precipitation, which makes large-scaleproduction feasible. BPMPC is a hydrophilic polymer due to the highconcentration of MPC, and has a high solubility in aqueous and alcoholsolutions. The butyl methacrylate component in the terpolymer aids inuniformity and substrate wetting (both hydrophobic and hydrophilic),along with providing additional photochemical cross-linking sites. Asdescribed above, the benzophenone component of BPMPC acts as across-linker between the hydrophilic polymer and any organic substratethrough C—H activation.

Scheme 1A shows synthesis of the BPMPC copolymer. Scheme 1B shows thechemical structure of SNAP and NO decomposition along with innocuousN-acetylpenicillamine byproduct.

The cross-linking kinetics of BPMPC was investigated by UV-visspectroscopy on isobutyltrichlorosilane (iBTS) functionalized quartzsubstrates. The polymer solution (10 μL, 10 mg mL⁻¹) was drop cast onalkylated quartz and the solvent allowed to evaporate. The UVcrosslinking reaction was monitored by UV-vis, where the decreasingabsorbance of the BP group at 255 nm occurs with increased irradiationtime. FIG. 2 shows the UV-vis spectra, where the absorbance maxima at255 nm decreased dramatically from 0 to 120 s, and after 240 s, nofurther absorbance change was observed, even after prolongedirradiation. This result demonstrates that BPMPC crosslinking occurswith rapid kinetics, and only a few seconds are needed to covalentlybond BPMPC to a variety of different substrates.

To further confirm the deposition and cross-linking of the BPMPCpolymer, FTIR was conducted on coated substrates. In the IR spectra(FIG. 3), absorption peaks of the carbonyl (1720 cm⁻¹) and PC groups(1240, 1080, and 970 cm⁻¹) were observed and assigned to the MPC units.The peak at (1650 cm⁻¹) represents the C═O stretch of BP ketone. Asignificant reduction of this peak after irradiation further supportsthe formation of a network polymer of covalent linkage between BP andsubstrate.

To test the stability and durability of the coating, the water contactangle of the BPMPC coated silicon samples were monitored for up to 14days. The coated substrates were immersed in PBS solution and stirred inan incubator at 37° C., subsequently rinsed with H2O and dried withnitrogen before measuring the water contact angle (FIG. 4). The initialstatic contact angle for the bare CarboSil substrate is about 110°. Asignificant decrease in contact angle was observed after coating withBPMPC, from 110° to 50°, and this value of contact angle was maintainedover a period of 14 days immersed in an agitated PBS solution, whichsuggests the BPMPC coating was covalent bonded to the substrates anddoes not delaminate under physiological conditions.

The control samples used to test NO release behavior were coated onlywith CarboSil (the same polymer used to incorporate SNAP) while the testsamples were coated with CarboSil and BPMPC. The samples were tested inlightly agitated conditions to simulate physiological conditions. Thesamples were tested for a period of two weeks to demonstrate sustainablerelease of NO from the combination of hydrophobic and hydrophilicpolymers.

A SNAP leaching study was conducted first to measure the retention ofSNAP in the control and test polymer films during the course of thestudy. Measurements were recorded every other day for 2 weeks of soakingin PBS (FIG. 5A). A high amount of SNAP retention in the polymersensures sustained release of NO from the polymer matrix and minimizesthe risks (if any) associated with SNAP leaching.⁴⁴ As seen in FIG. 5A,for the initial measurement (Day 0 on graph of FIG. 5A) of leachingafter one hour of storage in 37° C. in PBS, a loss of 0.39±0.06% and0.47±0.26% was recorded for the control and BPMPC-coated substrate,respectively. This initial higher leaching for the BPMPC-coatedsubstrate is likely due to the hydrophilicity of the surface. However,SNAP leaching is almost identical between the control and test samplesas supported by the data from 1 and 3 days of storage in 37° C. forBPMPC-coated test films (0.96±0.26% and 1.44±0.26% for day 1 and day 3,respectively) and control films (0.96±0.05% and 1.55±0.07% for day 1 andday 3, respectively).

This trend of lower leaching of the SNAP molecules from the test filmswas observed over a 14 day period. It is also to be noted that at nopoint during the 14-day period were the samples kept at a temperaturebelow 37° C. or in dry conditions. This was done to closely simulatephysiological conditions for a continuous duration. The leaching forboth the control and test samples remained very low (<3.5%) over theexperiment duration but it is worth noting here that despite theexpectation that the hydrophilic coating could cause a higher leachingof SNAP molecules from the NO donor containing polymer by attractingwater molecules to the polymer surface, this was not the case. This islikely due to the ultrathin nature of the coating, which influences theaqueous interface, but not the bulk of the polymer film.

NO release measurements of the control and test samples were alsocarried out for a period of 14 days (FIG. 5B). Measurements with aSievers chemiluminescence NO analyzer is the standard characterizationmethodology accepted for polymers that release NO.⁴⁵⁻⁴⁷ It measures NOrelease in real time via the measurement of voltage produced by thephotons on the reaction of NO with ozone. Samples were stored at aconstant temperature of 37° C. and in PBS to simulate physiologicalconditions.

The results indicated a general trend of higher NO release from the testsamples (SNAP-containing material coated with CarboSil and BPMPC)compared to the control samples (SNAP-containing material coated withonly CarboSil). Day 0 measurements indicate that the test samples had aflux of 7.75±3.26 (×10⁻¹⁰) mol cm⁻² min⁻¹ while control samples had aflux of 3.76±1.50 (×10⁻¹⁰) mol cm⁻² min⁻¹ (Table 1). This burst of NOrelease from test samples results from the hydrophilicity of the topcoatwhich attracts water molecules to the sample surface. Water molecules onthe surface can accommodate release of NO as SNAP is more soluble (andprone to S—N═O bond cleavage) in aqueous conditions. After a day ofstorage, the control samples show a sharp decrease in NO flux(0.34±0.03×10⁻¹⁰ mol cm⁻² min⁻¹). This is seen because of the initialloss in SNAP molecules on day 0 and inability to maintain a hydratedstate for day 1. In contrast, BPMPC-coated substrates show three timesthe NO flux at 1.02±0.02×10⁻¹⁰ mol cm⁻² min⁻¹. This difference in NOflux can result from the hydrophilic topcoat of test samples thatmaintains a hydrated surface layer, which facilitates the release ofmore NO. This trend of higher NO flux from test samples when compared tocontrol samples can be seen through the 14-day study in Table 1 and thegraph in FIG. 5B.

TABLE 1 Comparison of nitric oxide release kinetics between control andcoated samples 10% SNAP with only 10% SNAP with CarboSil CarboSiltopcoat and BPMPC topcoat (NO flux (×10⁻¹⁰ mol min⁻¹ (NO flux (×10⁻¹⁰mol min⁻¹ cm⁻²) cm⁻²) Day 0 3.759 ± 1.491 7.746 ± 3.263 Day 1 0.335 ±0.032 1.016 ± 0.198 Day 3 0.141 ± 0.023 0.706 ± 0.157 Day 5 0.110 ±0.045 0.395 ± 0.208 Day 7 0.105 ± 0.008 0.498 ± 0.173 Day 10 0.247 ±0.324 0.383 ± 0.040 Day 14 0.127 ± 0.035 0.380 ± 0.125

At the end of the 14-day study, test samples (0.38±0.13 (×10⁻¹⁰) molcm⁻² min¹) still release three times the NO flux compared to the controlsamples (0.13±0.03 (×10⁻¹⁰) mol cm⁻² min⁻¹). This propensity of higherrelease of NO from CarboSil top-coated with BPMPC along with thereduction in leaching of SNAP is very beneficial and combines thematerial properties of CarboSil (low SNAP leaching) with a higher,sustained release of NO due to the hydrophilic BPMPC topcoat.

As mentioned earlier, the BPMPC coating has excellent hydrophilicity,which helps inhibit the adsorption of proteins from solution. Fibrinogenand lysozyme were used as model proteins to evaluate the antifoulingproperties of the BPMPC coatings. Fibrinogen is a large (340 kD, pl=6.0)protein, and a key biomacromolecule in the coagulation cascade thatrapidly adsorbs to foreign surfaces and binds to and activatesplatelets. Lysozyme is a small protein (14 kD, pl=12) that is positivelycharged under physiological pH. FIG. 6A shows the adsorption thicknessincrease of Fibrinogen on CarboSil, CarboSil with 10% SNAP, BPMPC coatedCarboSil, and BPMPC coated CarboSil with 10% SNAP substratesrespectively. On the bare CarboSil films used as a control, thethickness increased about 2 nm after incubation for 24 hours, andincreased to over 30 nm after 2 weeks. The similar phenomenon wasobserved for CarboSil with 10% SNAP films, which indicated a high amountof protein adsorption on surface, and protein accumulation over time. Onthe other hand, for the CarboSil films coated with BPMPC, the adsorptionamount is significantly lower, only a 3 nm increase was observed afterincubation for 2 weeks. The large difference in adsorption thicknessconfirmed that BPMPC coating has excellent protein resistanceproperties, even after UV activation. As expected, the BPMPC coatedCarboSil with 10% SNAP films also shows low adsorption for Fibrinogen.Moreover, similar behavior was observed when films were subjected tolysozyme solution (FIG. 6B). The thickness increase in control group wasover 14 nm, while the coated group was less than 3 nm. The proteinadsorption results indicate that the hydrophilic BPMPC surface layerprovides excellent protein-resistant properties.

To further confirm the antifouling effectiveness of the durable BPMPCcoating, fluorescence microscopy was utilized to evaluate the proteinadsorption on the uncoated and coated CarboSil films using FITC labeledBSA protein. The fouling levels were compared between uncoated and BPMPCcoated CarboSil films using the same excitation light intensity andexposure time. FIG. 7A indicates protein adsorption on the controlsamples, and enhanced fluorescent signal (FIGS. 7B-7C) was observed inthe samples pretreated with BSA PBS solution. These results demonstratethat after incubation in protein solution, a large amount of BSA wasattached to the CarboSil samples, which facilitate the aggregation ofFITC-BSA. On the contrary, protein adhesion to the surface of BPMPCmodified samples was not observed (FIGS. 7D-7F), even after incubationin BSA solution for 7 days. From all of these results collectively, thecontrol films demonstrate large amounts of protein adsorption, while theBPMPC coated films display excellent antifouling properties.

Bacterial adhesion, which often results in biofilm formation, is aprevalent issue in moist and humid environments, including implanteddevices. The basic nutrients important for bacterial growth may beresourced from the device material, bodily proteins that attachpost-implantation, or other bodily macromolecular contaminants thatadhere to the surface of the device. Antimicrobial efficacy of thedesigned test samples was compared to the control samples to confirmtheir superior bactericidal and bacterial repulsion properties.

The samples were soaked in bacterial solutions containing ˜10⁶ CFU/mL ofS. aureus. S. aureus is a commonly found nosocomial infection bacteria.It has been increasingly linked with healthcare-associated infections inthe last two decades.⁴⁸ They are most commonly associated with cardiacdevices, intravascular catheters and urinary catheters, among otherprosthetic devices. This high prevalence of S. aureus along with itsknown affinity to proteins⁴⁹⁻⁵⁰ that foul medical devices has made it avery important pathogen used to evaluate the antimicrobial efficacy ofmedical device materials. For these reasons, bacterial adhesion study ofthe antifouling-biocide releasing polymer developed was done with S.aureus.

As mentioned in the introduction, the NO molecules liberated by thedecomposition of SNAP actively kill bacteria while the zwitteriontopcoat repels protein adsorption, leading to enhanced antimicrobialefficacy. After 24-hours of incubation, the antimicrobial effect of thetest samples was clearly observed. NO releasing polymers with a top-coatof BPMPC showed a bactericidal efficiency of 99.91±0.06% (˜3 logreduction, FIG. 8) compared to the control samples where a growth of˜10⁶ CFU/cm² was observed. This reduction is greater compared to filmswith only a BPMPC topcoat (70.15±14.13%) and also films with onlyNO-releasing moieties (98.88±0.54%). It can also be concluded from theresults that BPMPC alone only reduces bacteria adhesion. However,because NO is not a contact active antimicrobial but a diffusingbiocide, the SNAP-loaded samples also reduce bacterial adhesionsignificantly.

These results are consistent with the theoretical expectationsunderlying the surface chemistry of BPMPC and bactericidal properties ofNO. In summary, the synergistic effect of the modifiable NO-releasekinetics from CarboSil's surface and prevention of protein and/orbacterial adhesion due to BPMPC's surface chemistry will significantlyreduce undesired clinical consequences for implanted medical devices.

Conclusions

In conclusion, examples of the present disclosure have demonstrated acombination of NO release and BPMPC can produce a material withantimicrobial ability and excellent antifouling properties. Theformation of the covalent polymer network is rapid (less than 1 min)under mild UV conditions, and can be applied to various substrates, fromhydrophilic to hydrophobic. More importantly, even though the BPMPCcoating is around 50 nm, it resists moderate abrasion for over a weekwith retention of its antifouling property. Moreover, the NO releaseprofile indicated a higher NO release from the BPMPC coated sample whencompared to the control, with lower leaching of SNAP. The coatings werealso challenged with protein adsorption tests for an extended time (upto 2 weeks), where antifouling properties remain. It is noteworthy that,the high killing efficiency of SNAP to S. aureus is enhanced by BPMPCcoating. This one step photochemical attachment process of anantifouling coating to NO-releasing antimicrobial polyurethanes is asimple and scalable process that has application in both medical devicesand other industrial applications where antifouling and antimicrobialproperties are desired.

REFERENCES FOR EXAMPLE 1

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Example 2

The present example shows the combined effect of the NO-releasing donor(SNAP) and non-leaching quaternary ammonium (BPAM) to prevent theadherence of bacterial cells on the polymeric surface in addition tokilling bacteria beyond the direct point of contact. Briefly, polymericfilms were prepared by incorporating SNAP in CarboSil® 20 80A (a medicalgrade silicone-polycarbonate-urethane copolymer). The BPAM was surfaceimmobilized as a top-coat onto SNAP-CarboSil films by UV basedphotocrosslinking. The SNAP-BPAM based strategy to kill bacteria on thepolymer surface was characterized physically and chemically andvalidated for its antibacterial efficiency.

Materials and Methods

Materials

CarboSil® 20 80A thermoplastic silicone-polycarbonate-urethane(hereafter will be referred to as CarboSil) was obtained from DSMBiomedical (Berkeley, Calif.). N-Acetyl-D-penicillamine (NAP), methanol,sodium chloride, potassium chloride, potassium phosphate monobasic,sodium phosphate dibasic, dimethylacetamide (DMAc), tetrahydrofuran(THF), ethylenediaminetetraacetic acid (EDTA), and sulfuric acid wereobtained from Sigma-Aldrich (St. Louis, Mo.). N-Bromosuccinimide (NBS),2, 2′-azo-bis(2-methylpropionitrile) (AlBN), and N, N-dimethyl dodecylamine were purchased from Alfa-Aesar. LB broth, Lennox, and LB Agar,miller media were purchased from Fischer Bioreagents (Fair Lawn, N.J.).4-M ethylbenzophenone (Oxchem), cyclohexane (Honeywell), tert-amylalcohol (JT Baker), isopropyl alcohol (IPA) (JT Baker), sodium chloride(EMD Chemical), and peptone (HiMedia) were used without furtherpurification. Phosphate buffered saline (PBS), pH 7.4, containing 138 mMNaCl, 2.7 mM KCl, 10 mM sodium phosphate, was used for all in vitroexperiments including bacteria testing. The two-color fluorescentlive/dead BacLight bacterial viability kit L7012 (Molecular Probes, LifeTechnologies) which contains SYTO® 9 green fluorescent nucleic acidstain and the propidium iodide red fluorescent nucleic acid stain wasutilized to evaluate the bacterial viability. Gram-negative Pseudomonasaeruginosa (ATCC 27853) and Gram-positive Staphylococcus aureus (ATCC6538) were originally obtained from American Type Tissue Collection(ATCC). Instrumental Methods

UV-vis spectroscopy was performed on a Cary Bio Spectrophotometer(Varian). Two irradiation wavelengths, 254 and 365 nm, were utilized inthis study. The UV light sources were a Compact UV lamp (UVP) andFB-UBXL-1000 UV Crosslinker (Fisher Scientific) with bulbs of 254 nmwavelength for small (1×1 cm) and larger (2.5×2.5 cm) substrates,respectively. The substrates were held at 0.5 cm from the light sourceduring irradiation to obtain a power of 6.5 mW/cm². Another UV lightsource was an OmniCure, Series 1000 with 365 nm bandpass filter,equipped with a liquid-filled fiber optic waveguide. The polymericcomposite films were held 2 cm from the source for a power of 25 mW/cm².The thickness of the surface grafted BPAM film was measured using anM-2000 spectroscopic ellipsometer (J.A. Woollam Co., Inc). Water contactangles were measured by a DSA 100 drop shape analysis system (KRÜSS)with a computer-controlled liquid dispensing system. Water droplets witha volume of 1 μL were used to measure the static contact angle. Afluorescent microscope (EVOS FL, Thermo-Scientific) equipped with a 100×objective was used for live/dead bacterial viability photomicrographs. AGFP FITC filter cube (excitation: 490 nm, emission: 503 nm) was used forSYTO® 9 and a Texas Red filter cube (excitation: 577 nm, emission: 620nm) was used for the propidium iodide. The NO release study wasperformed using nitric oxide analyzer (NOA) 280i.

Synthesis of Bactericidal Agents and Fabrication of Polymeric Films

S-Nitroso-N-acetylpenicillamine (SNAP) synthesis—A method reported byChipinda et al. was modified for synthesizing SNAP from NAP [61].Briefly, sodium nitrite and NAP were added in an equimolar ratio to a1:1 mixture of methanol and water containing 2 M H₂SO₄ and 2 M HCl. Themixture was stirred in the dark (to prevent NO release by lightstimulation) for 40 min using a magnetic stirrer. Thereafter, thereaction vessel was placed in an ice bath to precipitate the SNAPcrystals. The resulting crystals were filtered out of the solution andallowed to air dry in dark followed by vacuuming to remove traces of anysolvent. SNAP crystals were stored in a freezer prior to use.

Benzophenone Based Antimicrobial Molecule (BPAM) Synthesis

The benzophenone based antimicrobial molecule (BPAM) was prepared usingthe previously reported procedure of Gao et al., [20]. Briefly,4-methylbenzophenone (6.0 g, 30.6 mM), NBS (6.0 g, 33.6 mM), AlBN (1.0g, 6.1 mM), and cyclohexane (100 mL) were added to a round-bottom flaskunder nitrogen atmosphere. The suspension was stirred under refluxovernight. After stirring, the mixture was cooled and filtered to removeany solid residues and the filtrate was concentrated under reducedpressure. The solid mixture was dissolved in diethyl ether and washedwith water, brine and dried over magnesium sulfate. The mixture wasfiltered and concentrated under reduced pressure. The recovered solidwas recrystallized from absolute ethanol to give fine white crystals.Yield: 7.1 g, 89%. ¹H NMR: δ, 7.80 (t, 2H, J=3.0 Hz); 7.78 (t, 2H, J=1.4Hz); 7.60 (t, 1H, J=7.0 Hz); 7.50 (d, 2H, 8.2 Hz); 7.49 (t, 2H, 7.6 Hz);4.53 (s, 2H). ¹³C NMR (CDCl₃): δ, 195.93, 142.09, 137.39, 132.54,130.52, 129.99, 128.92 128.33, 128.16, 32.25.

N-(4-benzoylbenzyl)-N,N-dimethylbutan-1-ammonium Iodide (BPAM)

(4-bromomethyl) benzophenone (1.7 g, 6.2 mM), N, N-dimethyldodecylamine(1.7 mL, 6.2 mM), and tert-amyl alcohol (5 mL) were added to a sealablepressure flask. The mixture was stirred and heated in the sealed vesselat 95° C. for 24 h. The flask was cooled to room temperature and thesolvent was removed under reduced pressure. The resulting brown waxysolid was recrystallized in hexane/ethyl acetate (7:4) to give a waxywhite solid. Yield: 1.7 g, 67%. ¹H NMR (CDCl₃): δ, 7.84 (dd, 4H, J=8.2,23.9 Hz); 7.75 (d, 2H, J=7.0 Hz); 7.59 (t, 1H, J=7.6 Hz); 7.47 (t, 2H,7.7 Hz); 3.57 (m, 2H); 3.35 (s, 6H); 1.80 (bs, 2H); 1.31 (bs, 4H); 1.21(bs, 16H); 0.84 (t, 3H, J=6.6 Hz). ¹³C NMR (CDCl₃): δ, 210.33, 139.75,136.70, 133.53, 133.24, 131.53, 130.54, 130.24, 120.66, 66.63, 64.12,49.85, 32.06, 29.69, 29.58, 29.46, 29.37, 26.42, 22.82, 14.29.

Fabrication of Antimicrobial SNAP-CarboSil Polymer Films

To begin making the polymeric SNAP films, 70 mg/mL of CarboSil wasdissolved in tetrahydrofuran (THF) as a solvent and stirred for 1 h atroom temperature using a magnetic stirrer. After complete dissolution,10% (w/w) of SNAP was quickly added and dissolved for 2 min in theCarbosil-THF solution. The SNAP films (10 wt %) were cast in Teflonmolds (diameter=2.5 cm) and dried overnight in dark to prevent undesiredloss of NO from the films using 3 ml of resulting SNAP-CarboSil-THFsolution. The films were coated twice with 50 mg/ml CarboSil solution(in THF). This outer coating ensures that SNAP does not leach out fromthe films and also generates a smooth surface. The control CarboSilfilms were prepared and coated in a similar manner, without the additionof SNAP.

Surface Immobilization of BPAM on SNAP-CarboSil Films

The BPAM film was immobilized onto SNAP-CarboSil film surfaces usingspray coating. BPAM/isopropanol solution (5 mg/mL) was sprayed using anairbrush spray gun from a distance of 20 cm onto a vertically placedsubstrate to achieve uniform coating. Upon solvent evaporation, a thinfilm of BPAM remained on the surface. Then BPAM coated films weresubsequently irradiated with UV light (254 nm, 6.5 mW/cm²) for 2 min tocovalently immobilize BPAM to the surface. The films were sonicated withisopropanol for 1 min to rinse off any residual, physisorbed BPAM anddried under a stream of nitrogen. FIG. 9 shows the fabrication procedureto make SNAP-BPAM CarboSil films (hereafter will be called SNAP-BPAMfilms).

Nitric Oxide Release Kinetics

Nitric oxide release from the SNAP and SNAP-BPAM films was measuredusing a Sievers Chemiluminescence Nitric Oxide Analyzer (NOA) 280i(Boulder, Colo.). The Sievers chemiluminescence NOA is considered as thegold standard for detecting in vitro nitric oxide release from thesubstrate. It is widely used for measurement of nitric oxide releasedfrom materials due to the ability to limit interfering species, such asnitrates and nitrites, as they are not transferred from the samplevessel to the reaction cell. Prior to NO release measurements, the films(n=3) were incubated for 1 h (PBS containing 100 mM EDTA, roomtemperature) to avoid the burst release associated with NO-releasingmaterials [42, 58]. Films were then placed in the sample vessel immersedin PBS (pH 7.4) containing 100 mM EDTA. Nitric oxide was continuouslypurged from the buffer and swept from the headspace using nitrogen sweepgas and a bubbler into the chemiluminescence detection chamber. Filmswere submerged in PBS with EDTA and stored in glass vials and kept at37° C. between NO-release measurements. Fresh PBS solution was used foreach NO-release measurement, and films were kept in fresh PBS solutionfor storage after each measurement.

UV-Vis Spectrophotometry

All UV-Vis spectra were recorded in the wavelength range of 200-700 nmusing a UV-Vis spectrophotometer Cary Bio Spectrophotometer (Varian) atroom temperature. CarboSil films and SNAP CarboSil films were developedon quartz glass substrates by spin coating with 200 μL of CarboSil/THFsolution (50 mg/mL) at 1000 rpm for 30 s. BPAM was top coated onCarboSil/quartz substrates by spray coating with BPAM/IPA solution (10mg/mL). The UV-vis spectrum of CarboSil was measured as the background.The presence of the S—NO group of SNAP provides characteristicabsorbance maxima at 340 and 590 nm [42, 62]. CarboSil films weredissolved in DMAc and absorbance values were measured at 340 nm. Theamount of SNAP was then determined using a calibration curve from knownmolar concentrations of SNAP in DMAC and compared to untreated controlCarboSil films.

Quantification of Adhered and Viable Colony Forming Units on PolymericSurface

In the example, the ability of the CarboSil polymer with SNAP-BPAMcombination to kill the adhered bacteria on the polymer surface wastested using the Gram-negative Pseudomonas aeruginosa and Gram-positiveStaphylococcus aureus bacteria which are amongst the most common causesof hospital-acquired infections (HAIs). A modified protocol of standardbacterial adhesion tests was used to quantify the viable colony formingunits of bacteria per surface area of the films (CFU/cm²) [63-65]. Asingle colony of bacteria was isolated from a previously culturedLB-agar plate and incubated in LB medium for 14 h at 37° C. at arotating speed of 150 rpm. The optical density of the culture wasmeasured at a wavelength of 600 nm (O.D₆₀₀) using UV-visspectrophotometer (Thermo scientific Genesys 10S UV-Vis) to ensure thatbacteria is in log phase of their growth. The bacteria culture was thencentrifuged at 3500 rpm for 7 min, the supernatant was discarded. Andsterile phosphate buffer saline (PBS), pH 7.4 was added to the bacterialpellet. This procedure was repeated twice to remove all traces of LBmedium and to suspend bacteria in PBS solution. In parallel, serialdilutions of the bacteria were prepared and plated in LB agar Petridishes in order to verify the consistency of concentration of viablecells between experiments. The OD₆₀₀ of the cell suspension in PBS wasmeasured and adjusted to the CFU/ml in the range of 10⁷-10⁹ based on thestandard calibration curve. CarboSil control, SNAP films, BPAM coatedCarboSil films and SNAP-BPAM films (n=3; surface area=0.94 cm²) wereexposed to bacterial cells (CFU: 10⁹-10⁷) at 37° C. for 24 h in a shakerincubator (150 rpm) after soaking them in PBS for 1 h to account for theburst effect. The 24 h incubation allows the bacteria to adhere to thesurface of the films and the adhered bacteria were acted upon by BPAMand NO. After 24 h, films were removed from the solution and any looselybound bacteria were washed by gently rinsing them with continuouslyflowing PBS (5 ml) using a pipette. The films were sonicated for 45 secusing an Omni-Tip homogenizer followed by vortexing for 30 sec tocollect the bound bacteria in 2 ml PBS solution. The PBS solution withbacteria was serially diluted (10⁻¹-10⁻⁵), plated in the solid LB agarmedium and incubated for 20 h at 37° C. After 20 h, the CFUs of theadhered viable bacteria on the surface of the polymer were countedkeeping in account the dilution factor.

Analysis of Residual NO Flux Post Bacteria Exposure

To ensure that fabricated film releases sufficient levels of nitricoxide after exposure to Pseudomonas aeruginosa or Staphylococcus aureusstrain for 24 h (Example 2: Quantification of adhered and viable colonyforming units on polymeric surface), the residual NO release wasconfirmed following the same procedure as explained in Example 2: NitricOxide release kinetics using the Sievers Nitric Oxide Analyzer (NOA).Triplicates of each film types (n=3) were analyzed for measuring theresidual NO flux.

Zone of Inhibition (ZOI) Analysis

A standard agar diffusion protocol was followed to conduct zone ofinhibition (ZOI) study to demonstrate the diffusive nature of NOmolecule released from SNAP and SNAP-BPAM films in the surrounding agar.This study was designed to prove that the NO release from the polymericcomposite can kill bacteria which are not in direct contact with thepolymeric films which BPAM alone fails to achieve. As a proof ofconcept, Gram-positive S. aureus and Gram-negative P. aeruginosabacteria strains were used for the study. A single colony of eachbacterium was suspended individually in LB and incubated at 37° C. for14 h at a rotating speed of 150 rpm. Using UV-Vis spectrophotometer(Genesis 10S-Thermo Scientific), the optical density (OD) of each of thebacterial cultures was measured at 600 nm (OD₆₀₀). The observed OD₆₀₀was adjusted to 1×10⁷ colony forming units per mL (CFUs/mL) based on acalibration curve based on the known concentration of S. aureus and P.aeruginosa. A sterile cotton swab was placed into each of the straincultures and then gently pressed and rotated against pre-made LB-agarPetri dishes (14 cm) to spread the bacteria aseptically and uniformly.Circular disks (diameter: 22 mm) of control, SNAP, BPAM, and SNAP-BPAMfilms were placed on top of bacterial culture and pressed gently. ThePetri dishes were incubated overnight at 37° C. in inverted position.The ZOI diameters were compared to evaluate the antimicrobial efficacyof NO releasing SNAP and SNAP-BPAM films.

Live/Dead Staining Assay

While the bacterial adhesion test allows counting the viable CFUs on thesurface of the polymer, it doesn't provide any quantitative orqualitative information on how many cells were initially attached anddied due to SNAP-BPAM biocidal activity. Using live/dead staining, weobserved both live and dead bacteria cells that were bound to thesurface of the polymer. This qualitative study was then combined withCell Profiler software to quantify the live and dead bacteria on thepolymeric surface. SYTO® 9 dye, yields green fluorescence and labels allbacteria in a population with intact membranes. In contrast, propidiumiodide, which yields red fluorescence, penetrates only the bacteria withdamaged membranes and replaces SYTO® 9 stains, causing a reduction ingreen fluorescence and the appearance of red fluorescence. Consequently,bacteria with damaged cell membranes can be distinguished from livebacteria. For each of the bacterial strains, 10 mL bacterial culture wasgrown to late log phase in broth (shaken at 100 rpm for 10 h at 37° C.).The culture was centrifuged at 4000 rpm for 10 min. The supernatant wasremoved and the pellet was suspended in sterile distilled water. Beforestaining, 10 μL bacterial suspension with a concentration around 10⁸CFU/mL was placed on the CarboSil, BPAM coated CarboSil, SNAP-blendedCarboSil, and SNAP-BPAM CarboSil films and dried at 37° C. for 5 min toachieve quick and intimate contact. Equal volumes of SYTO® 9 andpropidium iodide (1.5 μL) were combined, added to 1 mL of distilledwater, and mixed thoroughly. Diluted dye mixture (10 μL) was trappedbetween the slide with adhered bacteria and 18 mm square coverslip. Thesample was incubated in dark for 15 min at room temperature and imagedqualitatively with an EVOS fluorescence microscope. Both the live anddead bacteria from the fluorescent images were then quantified with CellProfiler software by randomly selecting different spots (n=3) on thefilms.

Statistical significance—For all the quantitative measurements n=3 datapoints were taken into consideration unless otherwise mentioned.Standard two-tailed t-test with unequal variance is used to do allstatistical comparisons. The data is reported as a mean±standarddeviation and the significance with a p-value <0.05 is stated forcomparisons.

Results

Photocrosslinking of BPAM on SNAP-CarboSil and Quantification of TotalSNAP

The benzophenone moiety of BPAM photochemically reacts with C—H groupsof the CarboSil polymer to form new C—C bonds at the interface. Uponabsorption of UV light, the promotion of one electron from a nonbindingn orbital to the antibonding π* orbital of the carbonyl group yields abiradicaloid triplet state where the electron-deficient oxygen n orbitalinteracts with surrounding weak C—H δ bonds, resulting in H abstractionto complete the half-filled n orbital. The two resulting carbon radicalsthen combine to form a new C—C bond. This process was monitored by thedecrease in absorbance of the n-π* transition of BP using UV-visspectrometry (FIG. 10A). Before exposure to UV light, the λ_(max)absorbance at 255 nm was observed, which is the characteristic n-π*transition of BPAM [20]. A spectrum shoulder ranging from 310-350 nm isassigned to the UV absorbance (λ_(max)) of SNAP [61]. The low intensityof the SNAP absorbance is due to the low concentration of SNAP withinthe CarboSil polymer matrix. After UV irradiation for 90 seconds,absorbance at 255 nm decreased, indicating the completion of thecrosslinking reaction. The broad shoulder at 250-300 nm could be due toslight photo-oxidation of the polycarbonate base [66]. The remainingSNAP content after UV treatment is shown in FIG. 10B and was confirmedto maintain 95.44±2.5% of the initial SNAP content. This can be mainlydue to the presence of a top coat of Carbosil in the SNAP films beforethe application of BPAM. In the past, the application of top coats ofCarboSil which are in the order of 100 microns has been shown tosignificantly reduce leaching when compared to non-top coated films[42]. These results confirmed that surface immobilized BPAM doesn'tadversely cause significant loss of SNAP from the polymeric composite.

Nitric Oxide Release from SNAP and SNAP-BPAM CarboSil Films

Incorporation of SNAP to CarboSil has shown to provide continuous andlocalized NO delivery to specific sites of interest [67, 68]. Theincorporation of SNAP in medical grade polymers have been shown to behemocompatible and possesses stability during long-term storage at roomtemperature and physiological conditions [60, 69, 70].

In this study, release rates of NO were measured at physiologicalconditions (pH 7.4, 37° C.) to demonstrate that the presence of the BPAMcoating does not adversely affect the NO release profile. The release ofNO from these compounds stems from the breaking of the S—NO bond thatcan be catalyzed using heat, light, moisture, or metal ions [42, 57,62]. Representative real-time NO release profiles from SNAP andSNAP-BPAM films was recorded via NOA as shown in FIG. 11. SNAP filmsexhibited an initial release rate of 1.35±0.11×10⁻¹⁰ mol min⁻¹ cm⁻² andrelease rate of 0.28±0.02×10⁻¹⁰ mol min⁻¹ cm⁻² after 24 h. SNAP filmswith BPAM top coat showed an increase in NO flux both at the initial(2.58±0.25×10⁻¹⁰ mol min⁻¹ cm⁻²) and at the 24-hour time point(0.59±0.04×10⁻¹⁰ mol min⁻¹ cm⁻²) (FIG. 12). This may be attributed tothe increase in hydrophilicity of the films with the presence of BPAMtopcoat as described above. The increased flux is still well within thephysiological range (0.5-4.0×10⁻¹⁰ mol min⁻¹cm⁻²) making it relevant andeffective for biomedical device applications [39]. The NO flux exhibitedby the films is sufficient to kill bacteria beyond 24 h. This has beenshown in vivo in a 7-day sheep catheter model using similar hydrophobicpolymers with 10 wt. % SNAP [71]. These materials exhibit similar NOrelease characteristics over a 28-d period and demonstrate that NOrelease rates at the lower end of physiological limits are stilleffective in providing antibacterial activity. Another report has alsoshown that hydrophobic polymers with SNAP have extended NO-release atphysiological levels (up to 20 days) [67]. Furthermore, theincorporation of SNAP in medical grade polymers are not onlyhemocompatible and biocompatible but also stable during long-termstorage (6 months) at room temperature and physiological conditions [60,69, 70].

Coating Thickness and Contact Angle Analysis

The thickness and static contact angle measurements are among the mostrelevant physical characterization for a polymeric coating. The resultsof this characterization for CarboSil films functionalized withindividual and combination of antibacterial agents are illustrated inTable 3.1. The thickness of the crosslinked BPAM coatings on CarboSiland SNAP CarboSil films after UV irradiation were 45.7±0.3 nm and47.9±0.5 nm, respectively, indicating successful grafting of a BPAMcoating. Water contact angles of control CarboSil, SNAP CarboSil, BPAMCarboSil, and SNAP-BPAM CarboSil surfaces are also listed in Table 1. Wehave previously shown that blending SNAP into the CarboSil polymericmatrix does not affect the hydrophobicity of the polymer [68]. The studyshowed that due to lower water uptake of CarboSil, limited leaching ofSNAP has been seen from SNAP doped CarboSil. Leaching is further reducedwith the use of a top coat (<10% of total SNAP loading in first 24 h) ascompared to the non-coated films. A change in the topcoat of Carbosil ismuch thicker than photo-crosslinked BPAM coat (100 μm vs 50 nm for BPAM)and hence little or no changes to the leaching kinetics of SNAP isexpected.

TABLE 2 Physical properties of antibacterial SNAP-BPAM film SNAP BPAMSNAP-BPAM Sample CarboSil CarboSil CarboSil CarboSil Thickness (nm) N/AN/A 45.7 ± 0.3 47.9 ± 0.5 Contact Angle (°) 119.3 ± 0.4 115 ± 0.2 67.9 ±0.6 63.5 ± 0.5 The data is reported as a mean ± standard deviation for n= 3 samples and the significance with a p-value < 0.05 is stated forcomparisons.

Blending SNAP into the CarboSil polymeric matrix does not affect thehydrophobicity of the polymer. The CarboSil control surface was found tobe hydrophobic with contact angle (CA) of 119.3±0.4°. The surfacegrafted layer of BPAM significantly decreases the hydrophobicity of theCarboSil based polymer film, reducing the CA to 63.5°±0.5°, resultingfrom the positively charged ammonium functional groups. The increase inthe surface hydrophilicity is expected to increase the antibacterialefficacy of the SNAP-BPAM polymer films as studies have shown a markedincrease in NO release from the hydrophilic surface when compared to thehydrophobic surfaces [42]. This is in line with the results obtainedfrom the NO release kinetics study. Furthermore, increasedhydrophilicity helps in the repulsion of non-specific proteinadsorption, and ultimately bacterial adhesion [72, 73] as confirmed bythe bacterial adhesion test and Live/Dead staining.

Quantification of Adhered Viable Bacteria (CFU/Cm²)

Biofilm formation is a major cause of morbidity and mortality associatedwith hospital acquired infection (HAIs). Staphylococcus aureus, aGram-positive bacterium, and Pseudomonas aeruginosa, a Gram-negativebacterium are among the most common causes of nosocomial bloodstreaminfections that can form embedded biofilm matrices on indwellingbiomedical devices [74-76]. As shown in FIG. 13, the amount of viable P.aeruginosa and S. aureus adhered on SNAP-BPAM film surfaces aresignificantly lower than that of control films. While BPAM and SNAP areexcellent antimicrobial agents, the combination offers severaladvantages not possessed by an individual antimicrobial agent. BPAM byitself has a superior antibacterial potential towards Gram-negative P.aureginosa as compared to SNAP, and SNAP is superior with respect to itsbactericidal action against Gram-positive S. aureus. The combination isvery effective against both Gram-positive and negative bacteria. Overallthe SNAP-BPAM films reduced the adhered viable bacteria (both Gramspositive and negative) to the maximum extent as compared to the controlfilms. SNAP-BPAM films showed a 4-log reduction for Gram-positive S.aureus and 3-log reduction for Gram-negative as compared to the CarboSilcontrol. FIG. 13 and Table 3 represents the graphs and the raw data forthe reduction in adhered CFU of both the bacteria per surface area ofthe polymeric composites. The difference in the results between the twobacteria can be attributed to the difference in the cell wall andmembrane composition of Gram-positive and Gram-negative bacteria [77].

TABLE 3 A comparative viable bacterial adhesion data for polymericcomposites and NO flux analysis before and after bacterial exposureResidual Residual NO flux* NO S. aureus P. aeruginosa NO flux* NO flux*Films (0 h) flux*(24 h) CFU/cm² CFU/cm² Post S. A Post P. A Carbosil — —2.1 × 10⁸ ± 3.0 × 10⁸ ± — 7.3 × 10⁶ 9.7 × 10⁵ BPAM — — 6.0 × 10⁵ ± 3.8 ×10⁵ ± — 5.9 × 10⁴ 6.2 × 10⁴ SNAP 1.35 ± 0.28 ± 6.6 × 10⁴ ± 1.3 × 10⁶ ±0.19 ± 0.30 ± 0.11 0.02 2.0 × 10⁴ 5.9 × 10⁵ 0.07 0.12 SNAP − 2.21 ± 0.35± 3.0 × 10⁴ ± 2.3 × 10⁵ ± 0.70 ± 0.92 ± BPAM 0.25 0.04 7.0 × 10³ 8.3 ×10⁴ 0.32 0.06 *NO flux (×10⁻¹⁰ molmin⁻¹cm⁻²). The data is reported as amean ± standard deviation for n = 3 samples and the significance with ap-value < 0.05 is stated for comparisons. Post S. A means NO flux after24 hours of S. aureus exposure and post P. A means NO flux after 24hours of P. aeruginosa exposure.

Non-leaching BPAM can only act on bacteria in intimate contact while NOcan act beyond direct point of contact because of diffusion. Theactivity of BPAM is also diminished with time by the layer of bacterialcells (live or dead) on polymeric composites as they tend to neutralizethe charge on quaternary ammonium. This problem can be addressed by theapplication of NO. The small molecular size of NO allows it to diffusethrough the bacterial biofilm and kill the bacterial cells which areotherwise resistant to bactericidal agents. In other words, thegradually released NO extended the life of BPAM by lowering theconcentration of surrounding bacteria near the surface. The SNAP-BPAMfilms have relatively higher hydrophilicity (due to BPAM coat) than SNAPfilms alone which increased the NO flux release from the SNAP-BPAMfilms.

The residual NO analysis after exposing films to bacteria suspension(FIG. 14) showed an abundance of NO up to 0.92±0.05×10⁻¹⁰ mol min⁻¹ cm⁻²flux, suggesting that these films can continue to exhibit antibacterialproperties beyond 24 h. The combined action of these bactericidal agentsvia multiple mechanisms of bacteria killing warrants a significantreduction in viable bacterial load for both Gram-positive and negativestrains.

Bacterial Killing Via NO Diffusion

While BPAM is an excellent antimicrobial agent, due to its non-diffusivenature, it cannot kill the bacteria protected within the biofilm matrix.Moreover, the charge density of surface-bound BPAM might be neutralizedwith anionic cellular components in the cytoplasm that is expelled outof the dead bacteria or screened by the layer of negatively charged deadbacterial cells covering the material's surface [26, 27]. Therefore, thediffusive nature of NO can be beneficial in biofilm eradication beyondthe close vicinity of the material. The standard agar diffusion testallowed us to show the bactericidal effect of the NO releasing films inthe presence and absence of the BPAM.

The CarboSil films with incorporated antimicrobial agents (SNAP-BPAM)and their combination resulted in a zone of inhibition (ZOI) ofdifferent diameters when exposed to LB agar plates with bacterialculture. As expected, the result demonstrated that BPAM has no ZOI,while the SNAP films and SNAP-BPAM films showed a clear ZOI due to therelease of NO gas from SNAP when placed in an incubator at 37° C. for 20h (FIGS. 15A-B). The explanation for this is the diffusive nature of NOthat can penetrate in the LB agar and hence prevent the bacterial growthin the area around the film. The breaking of the S—NO bond in SNAPcauses the release of NO. On the other hand, BPAM is non-diffusive innature and hence can only act on bacteria which are in direct contact.From an application point of view, this will be beneficial for biofilmeradication as NO due to its small size would easily penetrate throughthe matrix of a bacterial biofilm. Even though BPAM didn't show any ZOI,it did prevent the growth of bacteria in direct contact underneath thefilm. The ZOI for P. aeruginosa was observed to be 24 mm for SNAP filmsand 26 mm for SNAP-BPAM films. Similarly, the ZOI for S. aureus wasobserved to be 24 mm with SNAP films and 25 mm for SNAP-BPAM films.Overall SNAP-BPAM composites showed the largest ZOI for both S. aureusas well as P. aeruginosa strains among all the composites. The biggerZOI with SNAP-BPAM combination is due to increase in NO flux with BPAMtopcoat (1.35±0.11×10⁻¹⁰ in SNAP films vs 2.58±0.25×10⁻¹° mol min⁻¹ cm⁻²in SNAP-BPAM films) as observed by chemiluminescence NOA. FIGS. 15A-Bshow the comparative ZOI diameter among the films for both the bacterialstrains. The difference in antibacterial efficacy shown towards the twobacterial strains can be attributed to the membrane properties ofGram-positive and Gram-negative bacteria [77].

Analysis and Quantitation of Live/Dead Stain Test on CarboSil Films

As mentioned above, the bacterial adhesion test showed the reduction inadhered viable cells and zone of inhibition agar diffusion testdemonstrated the killing of bacteria through diffusion respectively.However, the relative number of live and dead bacteria on the films werenot evaluated by either of these tests. Therefore, the antibacterialactivity of the SNAP-BPAM hybrid CarboSil films was also evaluated usinga live/dead fluorescent stain assay which stains the live cells as greenand the dead cells as red. Fluorescent images of S. aureus cells wereexposed on control CarboSil, BPAM, SNAP, and SNAP-BPAM films. Thebacterial cell count for the live/dead assay was quantitativelyestimated at three randomly selected spots by using Cell Profilersoftware as recommended by published reports [78, 79]. On the controlfilms, 97.35±0.72% bacterial cells showed green fluorescence, evenlydistributed across the surface, and retained intact spherical shape,suggesting that the tested bacterial cells were viable. On BPAM coatedCarboSil films, 94.41±0.61% of the total bacterial cells were stainedred, indicating the cell membrane disruption caused by contact withsurface-bound quaternary ammonium. On SNAP films, 97.58±0.44% of thetotal bacteria showed red fluorescence indicating dead cells. In thecase of SNAP-BPAM CarboSil films, a 99.62±0.59% killing efficacy wasachieved, demonstrating that the hybrid method effectively enhances theantibacterial activity of the functionalized biocompatible polymermaterial. This enhancement in bactericidal activity of SNAP-BPAM ascompared to SNAP and BPAM films is in line with the viable bacteriaadhesion test as well as the zone of inhibition testing. Notably, thepattern of aggregation of bacterial cells on the surface of the film wasobserved to be different and dependent on the antibacterial agent. Thebacterial cells on control CarboSil and BPAM films have a regularpattern of bacterial cell distribution. On the other hand, the cellswere aggregated together on SNAP Carbosil films, possibly due to thehydrophobicity of the CarboSil surface which caused repulsion to thenegatively charged bacterial cells. On the SNAP-BPAM films, the deadcells dispersed across the surface which might be due to the relativelylower hydrophobicity (C.A=63.5°±0.5°) of the SNAP-BPAM films' surface ascompared to SNAP films (C.A=115°±0.2°) resulting from the positivelycharged ammonium functional groups. Overall, lived/dead stainingexperiment combined with Cell Profiler software further validated thatcombined NO and surface-bound quaternary ammonium can provide dualantibacterial activities and thus significantly enhance the biocidalactivity as compared to the individual agent. The authors suggestfurther in vitro testing and high resolution image analysis on bacterialaggregation pattern to validate this plausible theoretical explanation.

Discussion

In the present example, a NO donor molecule (SNAP) and a surfaceimmobilized benzophenone based antimicrobial molecule (BPAM) were usedin combination and their combined effect to reduce microbial adhesionand viability on a medical grade polymeric surface was evaluated. Sincethe λ_(max) of BPAM and SNAP are distinctly separated, this benefits thehybrid material in two ways: (1) BPAM can absorb photons efficiently forthe photoreaction even in the presence of SNAP; (2) Photo-degradation ofSNAP is limited in the cross-linking process since the irradiationwavelength is 254 nm for the maximum energy absorbance efficiency ofBPAM.

The antibacterial potential of SNAP-BPAM films was tested via (i)Bacterial Adhesion test (ii) Agar diffusion test (iii) Live/Deadstaining. Combined these three tests allowed to quantitatively assessthe bacterial adhesion (both viable and non-viable) and their subsequentkilling by NO and BPAM action. The antimicrobial properties of NO aredue to denaturation of enzymes, deamination of DNA and lipid oxidationin bacteria matrix [52]. On the other hand, BPAM kills the bacteria thatare in direct contact by damaging the bacterial cell membrane integritydue to electrostatic interactions [21, 22].

SNAP based NO releasing polymers have many desirable properties from atranslational perspective such as the long-term storage stability (6months), ease of sterilization, and extended NO release (>2 weeks)without negatively affecting the physical characteristics,biocompatibility, and hemocompatibility of the polymer [42, 59, 60].Similarly, the surface grafted BPAM has been reported to exhibitexcellent antimicrobial activity against Gram-positive and Gram-negativebacteria on instant contact due to high surface charge density of thedeposited BPAM thin film [20]. However, this is the first example thatcombines SNAP and BPAM together to demonstrate their antibacterialpotential.

The present example demonstrated that the SNAP-BPAM combination hasbetter antibacterial properties than SNAP or BPAM alone. BPAM is highlyregarded as a bactericidal agent, but, it can only act on bacteria thatare in direct contact. NO molecule through its diffusive nature allowsacting on bacteria that are beyond the direct contact. This property isuseful to act on biofilm matrix that otherwise prevents the penetrationof antibacterial agent and keeps the bacteria immune. BPAM however,imparts a relatively hydrophilic surface to SNAP-CarboSil as apparentfrom a decrease in the contact angle. Studies have shown higher NOrelease from the hydrophilic surface when compared to the hydrophobicsurfaces which in turn resulted in higher bacterial killing [42]. Thisis in line with the NO flux analysis as the SNAP-BPAM films showedhigher NO flux as compared to the SNAP films. Furthermore, a hydrophilicsurface helps in the repulsion of non-specific protein adsorption, andultimately bacterial adhesion [72, 73] as confirmed by the bacterialadhesion test and Live/Dead staining. From a translational perspective,a biomedical implant fabricated with a non-leaching, hydrophilic surfacewould be able to form a solvated, aqueous layer upon contact with bodyfluids and thus reduce bacterial adhesion [80]. In addition, NO also hasthe advantage in blood-contacting device applications of the localizedeffect of temporarily inhibiting the activation of platelets thatapproach the polymer surface [56].

In vitro characterization of the SNAP-BPAM containing CarboSil polymerin the present example showed that such polymeric composites can yieldbetter antibacterial effect as compared to SNAP or BPAM individually(FIGS. 14, 15A-15B). Their distinct but very effective mode ofbactericidal action assures that the bacteria that in contact with thepolymeric composites are attacked via multiple bactericidal mechanisms.The NO release with the SNAP-BPAM combination was shown to be higherthan with SNAP alone making their cooperation more effective in terms ofdiffusion of NO into the biofilm (FIGS. 11 and 12). TheseSNAP-BPAM-CarboSil composites continued to release NO flux in thephysiological range past the bacteria exposure for 24 h (FIG. 14). Thesustained release of diffusible NO also extended the duration oflocalized action of BPAM by lowering the concentration of surroundingviable bacteria near the polymer surface allowing BPAM to kill anybacteria in local contact.

Overall, the combined action of these bactericidal agents via distinctmechanisms warrants a significant reduction in viable bacterial load forboth Gram-positive and negative strains. Furthermore, the rapid actionof NO (half-life <5 sec) and non-specific lethal action of surface-boundBPAM via physical membrane disruption limit the development of resistantbacterial strains [47, 51, 53-55].

Conclusion

In the current example, a polymeric composite was fabricated by blendingSNAP in the CarboSil polymer and BPAM was surface grafted via UVphotocrosslinking and its ability to inhibit bacteria on the surface wastested both qualitatively and quantitatively. The SNAP-BPAM combinationwas more effective in maximizing the bacterial load on the surface ofthe polymeric composite as compared to SNAP or BPAM films individually.The bacterial adhesion test demonstrated that combination is equallyeffective in minimizing the adhered viable CFUs of both Gram-positiveand Gram-negative bacteria whereas SNAP was more effective against S.aureus and BPAM alone was more effective against P. aureginosa whentested alone. As demonstrated by the agar diffusion test diffusivenature of NO allowed to kill the bacteria beyond the direct point ofcontact which BPAM can't achieve alone. This is important for potentialapplication in biofilm eradication. The live/dead staining allowed toobserve that SNAP-BPAM combination has a higher number of attached deadbacteria (than live) as compared to the controls. BPAM coat alsoincreased the hydrophilicity and higher NO flux as compared to the SNAPfilms. In addition, NO based material can be used in blood-contactingdevice applications because it temporarily inhibits the activation ofplatelets on the polymer's surface which BPAM cannot [56]. Overall, allthese characteristics are ideal for controlling biomedical devicerelated infections, especially in preventing bacteria from developingantibiotic resistance due to the different killing mechanisms exhibitedby SNAP and BPAM. Such highly effective antimicrobial attributes offer anew paradigm in the fabrication of antimicrobial surfaces for variousmedical device applications.

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Example 3

To explore the covalent grafting of zwitterionic polymers to varioussubstrates ranging from hydrophilic to hydrophobic, we incorporated thebenzophenone (BP) chromophore, a photoactive tethering reagent, into thepolymeric backbone.¹⁹⁻²⁴ The BP group can produce a diradical underlow-intensity UV irradiation (350-365 nm) that abstracts an aliphatichydrogen from a neighboring C—H bond to form a new C—C bond, withoutintensive UV oxidative damage to the polymer or substrates.²⁰ Throughthis process, network polymer films can be grafted with excellentdurability to a broad selection of C—H containing materials andsurfaces, and has been used for many applications such asmicrofluidics,²⁵⁻²⁶ organic semiconductors,²⁷ redox polymers,²⁸⁻²⁹anti-icing polymers,³⁰ and biosensors.³¹⁻³²

Nitric oxide (NO) is known as a potent and nonspecific bactericidalagent due to its natural broad-spectrum antimicrobial properties withlow risk for promoting bacterial resistance.³³⁻³⁵ NO utilizes severalantimicrobial mechanisms including nitrosation of amines and thiols,lipid peroxidation, tyrosine nitration and DNA cleavage.³⁶ Major classesof current NO donors include organic nitrates, metal-NO complexes,N-nitrosamines, and S-nitrosothiols,³⁷ S-nitroso-N-acetylpenicillamine(SNAP), a commonly studied NO donor, exhibits significant antimicrobialand antithrombotic effects.³⁸⁻³⁹ In our previous studies, SNAP has beensuccessfully doped into CarboSil polymer films, and these SNAP-dopedpolyurethane-based materials can release NO for extended periods (20days) with very low levels of leaching.^(38, 40-41)

In this work, we synthesized zwitterionic terpolymers(2-methacryloyloxyethyl phosphorylcholine-co-butylmethacrylate-co-benzophenone, BPMPC) that can be covalently grafted toantimicrobial, NO-releasing CarboSil (silicone-polycarbonate-urethanethermoplastic) upon UV-irradiation. The polymer-coated surfaces arecharacterized in detail and the zwitterionic stability is assessed underphysiological conditions. The protein repellency properties of thesecoatings are evaluated. At the same time, no SNAP degradation wasobserved during coating or UV irradiation, and the release profileremained above the physiological level for 2 weeks with the zwitterionictop-coat. Moreover, enhanced antimicrobial activity was demonstratedwith bacteria testing.

Experiment Section

Materials

4-vinylbenzophenone (BP) was synthesized according to a previouslyreported method.³⁰ 2-Methacryloyloxyethyl phosphorylcholine (MPC),albumin from bovine serum (BSA), fluorescein isothiocyanate labeledbovine serum albumin (FTIC-BSA), N-acetyl-D-penicillamine (NAP), sodiumnitrite (NaNO₂), concentrated sulfuric acid (conc. H₂SO₄),tetrahydrofuran (THF), sodium phosphate monobasic (NaH₂PO₄), sodiumphosphate dibasic (Na₂HPO₄), potassium chloride, sodium chloride, andethylenediamine tetraacetic acid (EDTA) were purchased from SigmaAldrich (St. Louis, Mo.). 2,2′-azobis(2-methylpropionitrile) (AlBN) andn-butyl methacrylate (BMA) were bought from Alfa-Aesar (Haverhill,Mass.). Isobutyltrichlorosilane was purchased from Tokyo ChemicalIndustry (Portland, Oreg.). Concentrated hydrochloric acid (conc. HCl),sodium hydroxide (NaOH), and methanol were bought from Fisher-Scientific(Hampton, N.H.). Potassium phosphate monobasic (KH₂PO₄) and lysozymefrom egg white were purchased from BDH Chemicals—VWR International (WestChester, Pa.). CarboSil™ 20 80A UR STPU (referred to as CarboSil hereon)was acquired from DSM Biomedical Inc. (Berkeley, Calif.). Milli-Q filterwas used to obtain de-ionized (DI) water for all the aqueous solutionpreparations. Nitrogen and oxygen gas cylinders were purchased fromAirgas (Kennesaw, Ga.). Staphylococcus aureus (ATCC 6538, S. aureus) wasused for the bacterial experiments. LB Agar (LA), Miller and Luria broth(LB), Lennox were purchased from Fischer BioReagents (Fair Lawn, N.J.).All the chemicals were used without further purification.

In brief, CarboSil polymers with 10 wt % SNAP (test samples) and no SNAPcontent (control samples) were prepared using solvent evaporation and/orspin coating method. These samples were then coated with a zwitterioniccopolymer (referred to as BPMPC) which was covalently bonded to theCarboSil base polymers by UV-crosslinking. Surface analysis wasperformed on the films pre- and post-UV radiation to understand thecrosslinking behavior of the polyzwitterionic system. Test and controlsamples with the BPMPC coating were analyzed for their NO releasebehavior. The samples were then tested for protein adhesion for 14 daysin physiological conditions (37° C. in PBS) to evaluate antifoulingproperties of the topcoat. Finally, antimicrobial assay of the sampleswas done using a modified version of ASTM E2180 protocol.

Synthesis of NO Donor, SNAP

S-nitroso-N-acetylpenicillamine was synthesized using a revised approachfor a method previously reported.³⁸ 1M H₂SO₄ and 1M HCl were mixed withan equimolar amount of NAP, methanol and NaNO₂ aqueous solution. Thisreaction mixture was stirred for 20 minutes and then cooled for 7 hourswith a constant flow of air on the mixture. After evaporation of theunreacted portion of the reaction mixture, precipitated green crystalsof SNAP were filtered, collected and dried in a covered vacuumdesiccator. Dried crystals of SNAP were used for all experiments.

Synthesis of CarboSil Films Doped with SNAP

CarboSil films containing 10 wt % SNAP were prepared using solventevaporation method. 700 mg of CarboSil was dissolved in 10 mL of THF tomake the polymer solutions. 77 mg of SNAP was added to this solution fora final concentration of 10 wt % of SNAP. This polymer-SNAP blend wasstirred in dark conditions until the SNAP crystals dissolved completely.The blend was then transferred into Teflon molds and allowed to let thesolvent evaporate overnight in fume hood. The overnight dried films werethen cut into circular shapes of 0.8 cm diameter each. Each sample wasimmersed into a CarboSil solution without SNAP (40 mg mL⁻¹ of polymerconcentration in THF) to coat it (this was repeated thrice for eachsample). The samples were dried overnight and then dried under vacuumfor an additional 24 hours. This added drying time was included toeliminate any remaining THF which can affect any following studies.Weight of each film was recorded before the topcoat application for allSNAP leaching behavior tests. The formulated samples were stored in thefreezer (−18° C.) in the dark between experiments to prevent escape ofSNAP or consequent loss of NO. These SNAP-incorporated films were usedfor NO release, SNAP leaching and bacterial cell viability analyses. Allsamples used for the tests were less than a week old to ensure integrityof studies.

Synthesis of Zwitterionic Copolymer (BPMPC)

The polymer was synthesized by free radical polymerization. MPC (0.546g, 1.85 mmol), n-BMA (0.105 mL, 0.66 mmol) and BP (0.027 g, 0.132 mmol)were dissolved in 5.3 mL ethanol (total monomer concentration 1.0 mmolmL⁻¹) with initiator AlBN (0.01 mmol mL⁻¹) and the solution was pouredinto polymerization tube. After degassing with argon for 30 minutes, thepolymerization reaction was carried out under nitrogen flow at 60° C.for 16 h. The reaction was stopped by exposing the solution to air,cooled to room temperature, and poured into ethyl ether to precipitatethe polymer. The white solid was collected by vacuum filtration anddried under vacuum for 12 h. Yield: 0.552 g, 83%. ¹H NMR (D₂O) was takento confirm the polymer composition.

Crosslinking of BPMPC with Substrates

Silicon substrates were cut into 2.4 cm×2.4 cm pieces and sonicated withdeionized water, isopropanol, and acetone for 5 min each then driedunder nitrogen, followed by plasma (Harrick Plasma PDC-32G) clean andtreated with iBTS in toluene overnight before modification with thepolymer. CarboSil substrates were coated with polymer withoutpretreatment.

Two coating method were utilized when applying BPMPC on substrates: spincoating and spray coating. For spin coating, polymer modified film wasdeveloped on functionalized silicon substrate by using 0.5 mLBPMPC/ethanol solution (10 mg mL⁻¹) at 1000 rpm for 30 seconds. Spraycoating was applied for CarboSil films with and without SNAP.BPMPC/ethanol solution (2 mg mL⁻¹) was sprayed using a spray gun from adistance of 10 cm onto vertically placed substrates to achieve uniformcoating upon drying. We used spin coating in the protein adsorptionexperiments, and spray coating in SNAP/NO release and bacterialexperiments, based on method that afforded the smoothest, pin-hole freecoating on different forms of substrate. Then the BPMPC substrates wereirradiated with UV light (UVP, 254 nm, 6.5 mW cm⁻²) for 1 min tocovalently bond the BPMPC to the surface. The substrates were rinsedwith abundant ethanol to remove unattached BPMPC then dried undernitrogen.

Characterization of the Polymer Coatings

The surface wettability was characterized by measuring the static watercontact angle, which obtained from a DSA 100 drop shape analysis system(KRÜSS) with a computer-controlled liquid dispensing system. 1 μL DIwater droplets were deposited onto substrate surfaces, and the watercontact angles were measured within 10 seconds through the analysis ofphotographic images. The cross-linking kinetics of BPMPC coating wasinvestigated by a UV-vis spectroscopy (Varian) with 254 nm UV light. Thethickness of the spin-coated polymer layer on the silicon substrates andCarboSil substrates were measured by M-2000V Spectroscopic Ellipsometer(J.A. Woollam co., INC.) with a white light source at three incidentangles (65°, 70°, and 75). The thickness of the modified layer wasmeasured and calculated using a Cauchy layer model. Infraredspectroscopy studies of polymer coated films were done using aThermo-Nicolet model 6700 spectrometer equipped with a variable anglegrazing angle attenuated total reflection (GATR-ATR) accessory (HarrickScientific).

SNAP Leaching Study and NO-Release Profile

The percentage of SNAP discharged from the samples were quantified bynoting the absorbance of the PBS solutions (used to soak the samples) at340 nm (characteristic absorbance maxima of S—NO group of SNAP). Eachsample was weighed before coating with non-SNAP polymer solutions todetermine the initial amount of SNAP in each film. The films were thenimmersed in vials containing PBS (pH 7.4 with 100 μM EDTA to preventcatalysis of NO release by metal ions) and stored at 37° C. A UV-visspectrophotometer (Thermoscientific Genesys 10S UV-vis) was utilized toquantify the absorbance of the buffer solutions in the required timeintervals. The readings were converted to wt % of SNAP in the bufferutilizing the initial amount of SNAP present in each sample. 1 mLaliquots of the PBS solution in which the samples were soaked was usedfor each sample absorbance measurement to avoid any inconsistentreadings and three replicates were utilized for each quantification. Thecalibration graph with known amounts of SNAP in PBS (with EDTA) was usedto interpolate the absorbance quantifications recorded from the studyand convert them to concentrations of SNAP in the quantified sample.

SNAP incorporated in the polymers release NO in physiological conditionsand this release was measured and recorded in real time for the studyusing Sievers chemiluminescence NO analyzers® (NOA 280i, GE Analytical,Boulder, Colo., USA). The sample holder maintained dark conditions forthe samples to prevent catalysis of the NO production by any lightsource. It was filled with 5 mL of PBS (pH 7.4 with 100 μM of EDTA) tosoak the samples. EDTA acted as a chelating agent to prevent catalysisof NO production by metal ions in the PBS. This buffer solution wasmaintained at 37° C. by a temperature-regulated water jacket placedaround the sample holder. Once a baseline of NO flux without the sample(prepared according to Example 2: Nitric Oxide release kinetics) isestablished, the sample is then placed in the sample holder. Nitricoxide released by the sample in the sample holder was pushed and purgedtowards the analyzer by a continuous supply of nitrogen gas maintainedat a constant flow rate of 200 mL min⁻¹ through the sweep and bubbleflows. The NO released by the sample is pushed towards thechemiluminescence detection chamber.

The voltage signal produced is converted to concentration of NO anddisplayed on the analyzer's screen. Using the raw data in ppb form andNOA constant (mol ppb⁻¹ s⁻¹), the data in ppb is normalized for surfacearea of the sample and converted to NO flux units (×10⁻¹⁰ mol cm⁻²min⁻¹). Data was collected in the time intervals mentioned and sampleswere stored in a PBS (with EDTA) solution at 37° C. in dark conditionsbetween measurements. The PBS was replaced daily to avoid anyaccumulation of SNAP leached or NO released during the storage time. Theinstrument operating parameters were a cell pressure of 7.4 Torr, asupply pressure of 6.1 psig and a temperature of −12° C. Threereplicates were used for each measurement.

Protein Adhesion Assay

Protein adsorption test is a significant important method for evaluatingthe blood adhesion. Therefore, the thickness change of substrates beforeand after incubation in protein solutions was monitored, as anindication of protein adsorption. Coated substrates were incubated infibrinogen (1 mg mL⁻¹) and lysozyme (1 mg mL⁻¹) in PBS (pH 7.4, 0.01 M)solutions up to 14 days, followed by thickness measurement every day.

In the second approach, fluorescein isothiocyanate-bovine serum albumin(FITC-BSA, 2 mg mL⁻¹) in PBS solution was used to evaluate the proteinadsorption behavior on the surface of CarboSil substrate modified byBPMPC.⁴²⁻⁴³ Substrates were immersed in FITC-BSA solution for one andhalf hour at 37° C., then rinsed with distilled water and dried withnitrogen. The substrates with protein then analyzed by Nikon EclipseNI-U fluorescence microscope (Nikon Instruments, Inc.), using a 5×objective lens, with filter set (Ex/Em 470/525 nm). To confirm thelong-term resistance to protein adsorption, the substrates wereincubated in BSA (1 mg ml⁻¹) PBS solution for up to 7 days at 37° C.before putting in FITC-BSA solution.

Bacterial Assay

Bacterial adhesion for each of the samples was calculated in terms ofthe bacterial cell viability using serial dilution after an incubationperiod of 24 hours. The method used to perform this assay was based on amodified version of the American Society for Testing and Materials E2180protocol. S. aureus was used for antimicrobial evaluation of thesamples. Bacteria were cultured in LB Broth (Lennox) at 37° C. and grownto ˜10⁶ colony-forming units (CFU) per mL as measured by opticaldensity. The resulting overnight culture was collected by centrifugation(2500 g, 7 min) and resuspended in PBS. This resuspended bacterialsuspension was used for incubation of polymer samples for 24 hours.

After incubation with the bacterial solution, samples were washed gentlywith PBS to remove any unbound bacteria. The samples were then placed in1 mL of PBS and homogenized for 1 minute each to transfer any adheredbacteria to this new PBS solution. After homogenization, homogenatesamples were serially diluted and plated onto LB Agar nutrient plates(37° C.). Bacterial viability was determined by counting the colonies oneach plate manually. Calculation of bacterial adhesion was done bycounting number of colonies per cm² of each sample.

Statistical Analysis. All data are quantified as mean±standard deviationwith an n≥3 for all trials. The results between the control and testfilms were analyzed by a comparison of means using student's t-test.Values of p were obtained for the data analyzed and p<0.05 wasconsidered significant.

Results and Discussion

The zwitterionic polymer (BPMPC) was synthesized by radicalpolymerization in ethanol. The copolymer composition was confirmed by ¹HNMR spectroscopy, and consisted of 74:18:8 (MPC:nBMA:BP), which roughlymatched the monomer feed ratio. This ratio provided the optimalanti-fouling result (discussed below) along with the most uniformcoating on both hydrophobic and hydrophilic substrates. The polymersynthesis is simple and straightforward, no further purification isrequired besides precipitation, which makes large-scale productionfeasible. BPMPC is a hydrophilic polymer due to the high concentrationof MPC, and has a high solubility in aqueous and alcohol solutions. Thebutyl methacrylate component in the terpolymer aids in uniformity andsubstrate wetting (both hydrophobic and hydrophilic), along withproviding additional photochemical cross-linking sites. As describedabove, the benzophenone component of BPMPC acts as a cross-linkerbetween the hydrophilic polymer and any organic substrate through C—Hactivation.

The cross-linking kinetics of BPMPC was investigated by UV-visspectroscopy on isobutyltrichlorosilane (iBTS) functionalized quartzsubstrates. The polymer solution (10 μL, 10 mg mL⁻¹) was drop cast onalkylated quartz and the solvent allowed to evaporate. The UVcrosslinking reaction was monitored by UV-vis, where the decreasingabsorbance of the BP group at 255 nm occurs with increased irradiationtime. FIG. 2 shows the UV-vis spectra, where the absorbance maxima at255 nm decreased dramatically from 0 to 120 s, and after 240 s, nofurther absorbance change was observed, even after prolongedirradiation. This result demonstrates that BPMPC crosslinking occurswith rapid kinetics, and only a few seconds are needed to covalentlybond BPMPC to a variety of different substrates.

To further confirm the deposition and cross-linking of the BPMPCpolymer, FTIR was conducted on coated substrates. In the IR spectra,absorption peaks of the carbonyl (1720 cm⁻¹) and PC groups (1240, 1080,and 970 cm⁻¹) were observed and assigned to the MPC units. The peak at(1650 cm⁻¹) represents the C═O stretch of BP ketone. A significantreduction of this peak after irradiation further supports the formationof a network polymer of covalent linkage between BP and substrate.

To test the stability and durability of the coating, we monitored thewater contact angle of the BPMPC coated silicon samples up to 14 days.The coated substrates were immersed in PBS solution and stirred in anincubator at 37° C., subsequently rinsed with H₂O and dried withnitrogen before measuring the water contact angle (FIG. 4). The initialstatic contact angle for the bare CarboSil substrate is about 110°. Asignificant decrease in contact angle was observed after coating withBPMPC, from 110° to 50°, and this value of contact angle was maintainedover a period of 14 days immersed in an agitated PBS solution, whichsuggests the BPMPC coating was covalent bonded to the substrates anddoes not delaminate under physiological conditions.

The control samples used to test NO release behavior were coated onlywith CarboSil (the same polymer used to incorporate SNAP) while the testsamples were coated with CarboSil and BPMPC. The samples were tested inlightly agitated conditions to simulate physiological conditions. Thesamples were tested for a period of two weeks to demonstrate sustainablerelease of NO from the combination of hydrophobic and hydrophilicpolymers.

A SNAP leaching study was conducted first to measure the retention ofSNAP in the control and test polymer films during the course of thestudy. Measurements were recorded every other day for 2 weeks of soakingin PBS (FIG. 5A). A high amount of SNAP retention in the polymersensures sustained release of NO from the polymer matrix and minimizesthe risks (if any) associated with SNAP leaching.⁴⁴ As seen in FIG. 5A,for the initial measurement (Day 0 on graph of FIG. 5A) of leachingafter one hour of storage in 37° C. in PBS, a loss of 0.39±0.06% and0.47±0.26% was recorded for the control and BPMPC-coated substrate,respectively. This initial higher leaching for the BPMPC-coatedsubstrate is likely due to the hydrophilicity of the surface. However,SNAP leaching is almost identical between the control and test samplesas supported by the data from 1 and 3 days of storage in 37° C. forBPMPC-coated test films (0.96±0.26% and 1.44±0.26% for day 1 and day 3,respectively) and control films (0.96±0.05% and 1.55±0.07% for day 1 andday 3, respectively).

This trend of lower leaching of the SNAP molecules from the test filmswas observed over a 14 day period. It is also to be noted that at nopoint during the 14-day period were the samples kept at a temperaturebelow 37° C. or in dry conditions. This was done to closely simulatephysiological conditions for a continuous duration. The leaching forboth the control and test samples remained very low (<3.5%) over theexperiment duration but it is worth noting here that despite theexpectation that the hydrophilic coating could cause a higher leachingof SNAP molecules from the NO donor containing polymer by attractingwater molecules to the polymer surface, this was not the case. This islikely due to the ultrathin nature of the coating, which influences theaqueous interface, but not the bulk of the polymer film.

NO release measurements of the control and test samples were alsocarried out for a period of 14 days (FIG. 5B). Measurements with aSievers chemiluminescence NO analyzer is the standard characterizationmethodology accepted for polymers that release NO.⁴⁵⁻⁴⁷ It measures NOrelease in real time via the measurement of voltage produced by thephotons on the reaction of NO with ozone. In this study, samples werestored at a constant temperature of 37° C. and in PBS to simulatephysiological conditions.

The results indicated a general trend of higher NO release from the testsamples (SNAP-containing material coated with CarboSil and BPMPC)compared to the control samples (SNAP-containing material coated withonly CarboSil). Day 0 measurements indicate that the test samples had aflux of 7.75±3.26 (×10⁻¹⁰) mol cm⁻² min⁻¹ while control samples had aflux of 3.76±1.50 (×10⁻¹⁰) mol cm⁻² min⁻¹ (Table 4). This burst of NOrelease from test samples results from the hydrophilicity of the topcoatwhich attracts water molecules to the sample surface. Water molecules onthe surface can accommodate release of NO as SNAP is more soluble (andprone to S—N═O bond cleavage) in aqueous conditions. After a day ofstorage, the control samples show a sharp decrease in NO flux(0.34±0.03×10⁻¹⁰ mol cm⁻² min⁻¹). This is seen because of the initialloss in SNAP molecules on day 0 and inability to maintain a hydratedstate for day 1. In contrast, BPMPC-coated substrates show three timesthe NO flux at 1.02±0.02×10⁻¹⁰ mol cm⁻² min⁻¹. This difference in NOflux can result from the hydrophilic topcoat of test samples thatmaintains a hydrated surface layer, which facilitates the release ofmore NO. This trend of higher NO flux from test samples when compared tocontrol samples can be seen through the 14-day study in Table 4 and thegraph in FIG. 5B.

TABLE 4 Comparison of nitric oxide release kinetics between control andcoated samples 10% SNAP with only 10% SNAP with CarboSil CarboSiltopcoat and BPMPC topcoat (NO flux (×10⁻¹⁰ mol min⁻¹ (NO flux (×10⁻¹⁰mol min⁻¹ cm⁻²) cm⁻²) Day 0 3.759 ± 1.491 7.746 ± 3.263 Day 1 0.335 ±0.032 1.016 ± 0.198 Day 3 0.141 ± 0.023 0.706 ± 0.157 Day 5 0.110 ±0.045 0.395 ± 0.208 Day 7 0.105 ± 0.008 0.498 ± 0.173 Day 10 0.247 ±0.324 0.383 ± 0.040 Day 14 0.127 ± 0.035 0.380 ± 0.125

At the end of the 14-day study, test samples (0.38±0.13 (×10⁻¹⁰) molcm⁻² min⁻¹) still release three times the NO flux compared to thecontrol samples (0.13±0.03 (×10⁻¹⁰) mol cm⁻² min⁻¹). This propensity ofhigher release of NO from CarboSil top-coated with BPMPC along with thereduction in leaching of SNAP is very beneficial and combines thematerial properties of CarboSil (low SNAP leaching) with a higher,sustained release of NO due to the hydrophilic BPMPC topcoat.

As mentioned earlier, the BPMPC coating has excellent hydrophilicity,which helps inhibit the adsorption of proteins from solution. Fibrinogenand lysozyme were used as model proteins to evaluate the antifoulingproperties of the BPMPC coatings. Fibrinogen is a large (340 kD, pl=6.0)protein, and a key biomacromolecule in the coagulation cascade thatrapidly adsorbs to foreign surfaces and binds to and activatesplatelets. Lysozyme is a small protein (14 kD, pl=12) that is positivelycharged under physiological pH. FIG. 6A shows the adsorption thicknessincrease of Fibrinogen on CarboSil, CarboSil with 10% SNAP, BPMPC coatedCarboSil, and BPMPC coated CarboSil with 10% SNAP substratesrespectively. On the bare CarboSil films used as a control, thethickness increased about 3 nm after incubation for 24 hours, andincreased over 30 nm after 2 weeks. The similar phenomenon was observedfor CarboSil with 10% SNAP films, which indicated a high amount ofprotein adsorption on surface, and protein accumulation over time. Onthe other hand, for the CarboSil films coated with BPMPC, the adsorptionamount is significantly lower, only a 2 nm increase was observed afterincubation for 2 weeks. The large difference in adsorption thicknessconfirmed that BPMPC coating has an excellent protein resistanceproperties, even after UV activation. As expected, the BPMPC coatedCarboSil with 10% SNAP films also shows low adsorption for Fibrinogen.Moreover, similar behavior was observed when films were subjected tolysozyme solution (FIG. 6B). The thickness increase in control group wasover 14 nm, while the coated group was less than 3 nm. The proteinadsorption results indicate that the hydrophilic BPMPC surface layerprovides excellent protein-resistant properties.

To further confirm the antifouling effectiveness of the durable BPMPCcoating, fluorescence microscopy was utilized to evaluate the proteinadsorption on the uncoated and coated CarboSil films using FITC labeledBSA protein. The fouling levels were compared between uncoated and BPMPCcoated CarboSil films using the same excitation light intensity andexposure time. The results indicated protein adsorption on the controlsamples, and enhanced fluorescent signal was observed in the samplespretreated with BSA PBS solution. These results demonstrate that afterincubation in protein solution, a large amount of BSA was attached tothe CarboSil samples, which facilitate the aggregation of FITC-BSA. Onthe contrary, protein adhesion to the surface of BPMPC modified sampleswas not observed, even after incubation in BSA solution for 7 days. Fromall of these results collectively, the control films demonstrate largeamounts of protein adsorption, while the BPMPC coated films displayexcellent antifouling properties.

Bacterial adhesion, which often results in biofilm formation, is aprevalent issue in moist and humid environments, including implanteddevices. The basic nutrients important for bacterial growth may beresourced from the device material, bodily proteins that attachpost-implantation, or other bodily macromolecular contaminants thatadhere to the surface of the device. Antimicrobial efficacy of thedesigned test samples was compared to the control samples to confirmtheir superior bactericidal and bacterial repulsion properties.

The samples were soaked in bacterial solutions containing ˜10⁶ CFU/mL ofS. aureus. S. aureus is a commonly found nosocomial infection bacteria.It has been increasingly linked with healthcare-associated infections inthe last two decades.⁴⁸ They are most commonly associated with cardiacdevices, intravascular catheters and urinary catheters, among otherprosthetic devices. This high prevalence of S. aureus along with itsknown affinity to proteins⁴⁹⁻⁵⁰ that foul medical devices has made it avery important pathogen used to evaluate the antimicrobial efficacy ofmedical device materials. For these reasons, bacterial adhesion study ofthe antifouling-biocide releasing polymer developed was done with S.aureus.

As mentioned in the introduction, the NO molecules liberated by thedecomposition of SNAP actively kill bacteria while the zwitteriontopcoat repels protein adsorption, leading to enhanced antimicrobialefficacy. After 24-hours of incubation, the antimicrobial effect of thetest samples was clearly observed. NO releasing polymers with a top-coatof BPMPC showed a bactericidal efficiency of 99.91±0.06% (˜3 logreduction, FIG. 8) compared to the control samples where a growth of˜10⁶ CFU/cm² was observed. This reduction is greater compared to filmswith only a BPMPC topcoat (70.15±14.13%) and also films with onlyNO-releasing moieties (98.88±0.54%). It can also be concluded from theresults that BPMPC alone only reduces bacteria adhesion. However,because NO is not a contact active antimicrobial but a diffusingbiocide, the SNAP-loaded samples also reduce bacterial adhesionsignificantly.

These results are consistent with the theoretical expectationsunderlying the surface chemistry of BPMPC and bactericidal properties ofNO. In summary, the synergistic effect of the modifiable NO-releasekinetics from CarboSil's surface and prevention of protein and/orbacterial adhesion due to BPMPC's surface chemistry will significantlyreduce undesired clinical consequences for implanted medical devices.

Conclusions

In conclusion, we have demonstrated a combination of NO release andBPMPC can produce a material with antimicrobial ability and excellentantifouling properties. The formation of the covalent polymer network israpid (less than 1 min) under mild UV conditions, and can be applied tovarious substrates, from hydrophilic to hydrophobic. More importantly,even though the BPMPC coating is around 50 nm, it resists moderateabrasion for over a week with retention of its antifouling property.Moreover, the NO release profile indicated a higher NO release from theBPMPC coated sample when compared to the control, with lower leaching ofSNAP. The coatings were also challenged with protein adsorption testsfor an extended time (up to 2 weeks), where antifouling propertiesremain. It is noteworthy that, the high killing efficiency of SNAP to S.aureus is enhanced by BPMPC coating. This one step photochemicalattachment process of an antifouling coating to NO-releasingantimicrobial polyurethanes is a simple and scalable process that hasapplication in both medical devices and other industrial applicationswhere antifouling and antimicrobial properties are desired.

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Example 4

Materials and Methods

Synthesis of SIM Material

Silicone films were first fabricated by mixing Sylgard 184 base tocuring agent (ratio of 10:1). The solution was cast into Teflon moldsand placed under vacuum for degassing. The casted solution was thenplaced in an oven (80° C., 90 min) for curing. Surface modification wasperformed in steps, illustrated in FIG. 16, as referred to below. Tocreate a hydroxyl group functionalized surface (step A), the siliconefilms were submerged in a mixture of 13 N HCl:30 wt. % H₂O₂ (50:50) inH₂O under mild agitation (15 min). The surfaces were then rinsed with DIH₂O and dried under vacuum. The amine functionalization (step B) wasthen achieved by submerging the hydroxyl-functionalized surfaces in 5wt. % APTMES in extra dry acetone for 2 h. Films were then rinsed withextra dry acetone to remove any non-covalently attached silane from thesurface, and vacuum dried for 24 h. Branching of the immobilizedmoieties was achieved through incubation of the amine functionalizedsurface in 2:1 (v/v) methanol:methyl acrylate (24 h) (steps E and I)followed by 2:1 (v/v) methanol:ethylenediamine (24 h) (steps F and J) asshown in FIG. 16. Samples were rinsed twice with methanol (20 mL)between incubating solutions. Amine-functionalized surfaces were thensubmerged in 10 mg mL⁻¹ NAP-thiolactone in toluene for 24 h (steps C, G,and K), allowing for the ring opening reaction of thiolactone to bind tofree amines.^(24, 25) The samples were then air-dried for 5 h tocompletely remove any residual solvent. Nitrosation of the immobilizedNAP (steps D, H, and L) was achieved by incubation in neat tert-Butylnitrite for 2 h. The resultant SIMS samples were stored at −20° C. forfurther experiments.

Contact Angle and FTIR Analysis

Surface properties and proof of attachment of nitric oxide donors tosilicone surfaces was analyzed using contact angle measurements andFTIR. Static contact angle was measured using a DSA 100 drop shapeanalysis system (KRU{umlaut over ( )}SS) with a computer-controlledliquid dispensing system (Krüss). A 3 μL droplet of water was placed onvarious silicone films, and the average of left and right contact angleswere measured via the Krüss software. Infrared spectroscopy studies ofthe samples were done using a Thermo-Nicolet model 6700 spectrometer.

Free Amine Quantification

Quantification of APTMES attachment to the surface of the PDMS was doneusing an ATTO-TAG (3-2-(furoyl quinoline-2-carboxaldehyde)) (FQ)Amine-derivatization kit (ThermoFisher Scientific, Waltham, Mass.). Eachprimary amine forms a conjugate with the FQ reagent which can then befluorescently detected. Fluorescent count was measured using a BiotekSynergy microplate reader (Winooski, Vt.). The tested samples of APTMESfunctionalized PDMS were first carefully measured before being placed ina solution containing 15 μL of 10 mM potassium cyanide (KCN), 25 μL of0.01 M PBS (pH=7.4), and 10 μL of 10 mM FQ solution (in methanol). Thesamples were then protected from light and allowed to react for 1 h. Thesolutions containing the samples were then gently agitated to ensure allof the florescent product is removed from the surface. The sample pieceswere then discarded and the solutions with the fluorescent product wereplaced in a 96-well plate to be measured at excitation and emissionwavelengths of 480 nm and 590 nm, respectively. Calibration curves weredone using prepared using glycine to directly relate the fluorescentcount with primary amine concentration.

Nitric Oxide Release Characteristics

Nitric oxide release from the films containing SNAP was measured using aSievers Chemiluminescence Nitric Oxide Analyzer (NOA) 280i (Boulder,Colo.). The Sievers chemiluminescence Nitric Oxide analyzer isconsidered as the gold standard for detecting nitric oxide and is widelyused due to its ability to limit interfering species, such as nitratesand nitrites, as they are not transferred from the sample vessel to thereaction cell. Films were then placed in the sample vessel immersed inPBS (pH 7.4) containing 100 mM EDTA. Nitric oxide was continuouslypurged from the buffer and swept from the headspace using nitrogen sweepgas and bubbler into the chemiluminescence detection chamber.

Protein Repulsion Quantification:

Levels of protein adhesion were quantified for the various materialsusing a modified version of a previously reported method.⁷ FITC-humanfibrinogen (13 mg/mL, Molecular Innovations) was diluted to achieve 2 mgmL⁻¹ in PBS (pH 7.4). Silicone disks were incubated at 37° C. for 30 minin a 96-well plate, followed by the addition of the stock proteinsolution to achieve a concentration of 2 mg mL⁻¹.⁷ Following 2 h ofincubation, infinite dilution of the well contents was carried out towash away the bulk and any loosely bound protein from the materials. Thefluorescence of each well (n=8) was then measured using a 96-well platereader (Biotek), and the amount of protein adsorbed was determined via acalibration curve.

Bacterial Adhesion Assay:

The ability of the samples to inhibit growth and promote killing of theadhered bacteria on the polymer surface was tested following guidelinesbased on American Society for Testing and Materials E2180 protocol withthe commonly found nosocomial pathogen, Gram-positive S. aureus (ATCC6538). A single colony of bacteria was isolated from a previouslycultured LB-agar plate and incubated in LB Broth (37° C., 150 rpm, 14-16h). The optical density of the culture was measured at a wavelength of600 nm using a UV-vis spectrophotometer (Thermoscientific Genesys 10SUV-Vis) to ensure the presence of ˜10⁶-10⁸ CFU mL⁻¹. The overnightculture was then centrifuged at 2500 rpm for 7 min to obtain thebacterial pellet. The bacterial pellet obtained was resuspended insterile PBS. The polymer samples (SR control, SIM-N1, SIM-S1, SIM-N2 andSIM-S2) were then incubated in the bacterial suspension (37° C., 24 h,200 rpm). After incubation, samples were removed from the bacterialsuspension and rinsed with sterile PBS to remove any unbound bacteria.Each sample was then sonicated for 1 min using an Omni Tip homogenizerto collect adhered bacteria in sterile PBS. To ensure properhomogenization of the collected bacteria, the samples were vortexed for45 s each. The solutions were serially diluted, plated on LB agar mediumand incubated at 37° C. After 24 h, the total CFUs for serially dilutedand plated bacterial solutions were counted.

Platelet Adhesion Assay:

Freshly drawn porcine blood was purchased from Lampire Biologicals. Theanticoagulated blood was centrifuged (1100 rpm, 12 min) using theEppendorf Centrifuge 5702. The platelet rich plasma (PRP) portion wascollected carefully with a pipet as to not disturb the buffy coat. Theremaining samples were then centrifuged (4000 rpm, 20 min) to retrieveplatelet poor plasma (PPP). Total platelet counts in both PRP and PPPfractions were determined using a hemocytometer (Fisher). The PRP andPPP were combined in a ratio to give a final platelet concentration ca.2×10⁸ platelets mL⁻¹. Calcium chloride (CaCl₂) was added to the finalplatelet solution to achieve a final concentration of 2.5 mM.⁷ Disks ofeach respective surface were placed in a 5 mL blood tube. Approximately4 mL of the calcified PRP was added to each tube and incubated (37° C.,90 min) with mild rocking (25 rpm). Following the incubation, the tubeswere infinitely diluted with normal saline. The degree of plateletadhesion was determined using the lactate dehydrogenase (LDH) releasedwhen the adherent platelets were lysed with a Triton-PBS buffer using aRoche Cytotoxicity Detection Kit (LDH). The silicone disks were thenincubated in 1 mL of Triton-PBS buffer. After 25 min, 100 μL wastransferred to a 96-well plate and combined with 100 μL of the LDHreagent buffer per the supplier specifications. The absorbance of eachwell (duplicates of n=6) was then measured using a 96-well plate reader(Biotek), and the number of platelets adhered was determined using thecalibration curve.

Results and Discussion

To take advantage of the antimicrobial and platelet activationprevention properties of nitric oxide (NO), NO-donors (e.g.s-nitrosothiols or diazeniumdiolates) have been developed to providestorage and localized delivery of NO. Such NO-donors are adaptable forincorporation into polymeric materials typically used for medicaldevices, such as polyurethanes, silicones, or polyvinylchloride._(26, 27) The addition of these donors at various levels alsoprovides the ability to control the level of NO that is delivered fromthe materials.₂₈ NO-releasing materials have been used for reducingthrombus formation and bacterial growth during catheterization. However,once all or most of the NO has been released, the antibacterialproperties of the materials are significantly diminished. Thus, thepresent example provides examples of coatings and coated surfaces thatprovide both passive and active mechanisms of inhibiting thrombosis,bacterial growth, and surface fouling.

The present example demonstrates the ability of a material to not onlyoffer an increase in steric ability to prevent bacterial and proteinadhesion through a passive mechanism but also utilize the activebiocidal mechanism of NO. In addition to combining these mechanisms, theantifouling capability of the material is retained after all NO has beenreleased from the surface, making it attractive for long termapplications. Specifically, this is done by the immobilization of theNO-donor precursor N-acetyl-D-penicillamine (NAP) to silicone surfacesusing an alkylamine spacer (FIG. 16). As shown in FIG. 16, increasingthe grafting density of free amines was done through branching of theinitial spacer (steps E, F, I, J of FIG. 16) Following immobilization ofthe NO-donor precursor (steps C, G, K of FIG. 16), the grafted donor isnitrosated to its NO-rich form S-nitroso-N-acetyl-D-penicillamine (SNAP)(steps, D, H, L of FIG. 16).

SIM-N1 and SIM-S1 correspond to unbranched polymers that arenon-nitrosated (product of C on FIG. 16) and nitrosated (product of D onFIG. 16), respectively. SIM-N2 and SIM-S2 correspond to branchedpolymers that are non-nitrosated (product of G on FIG. 16) andnitrosated (product of H on FIG. 16), respectively. Finally, SIM-N4 andSIM-S4 correspond to branched polymers that are non-nitrosated (productof K on FIG. 16) and nitrosated (product of L on FIG. 16), respectively.FIG. 18 provides a schematic illustration of the branched (SIM-S2 andSIM-S4) and unbranched (SIM-S1) functionalized surfaces. In the presentexample, these materials will be referred to as abbreviated above forall the experiments, in addition to the bare silicone surface which willbe considered as the control. To ensure covalent bonding of the surfacemodifications, FTIR measurements were carried out (FIG. 17). Sincenitrogen atoms overlap in terms of FTIR peaks, appearance anddisappearance of amide and primary amines was seen as the reaction stepswere completed. This was followed by measurement of water contact angleto check for any significant differences in hydrophilicity of thefunctionalized surface. It is interesting to note here thathydrophilicity of the surfaces increased with increased NO-release(discussed in greater detail below). This could be attributed to loweravailability of amine functionalized surfaces as the reaction is morecomplete.

Table 8 Shows the Ability of Various Surface Modified SR Substrates toReduce Nonspecific Protein Adsorption Over 2 h

As seen in the design strategy, the NO-load and release capacity of thematerials was varied by branching of the initial alkyl spacer toincrease the number of free amines. This variation in NO-load andrelease capacity was measured by using a chemiluminescence nitric oxideanalyzer (NOA). NOA is the gold standard for measurement of NO flux frommaterials and is a very efficient and sensitive instrument that cananalyze NO release down to 1/10_(th) of ppb.₂₉ To ensure NO release wasonly from the surface functionalization and not from the bulk materialdue to possible swelling of the diamine group during the reactionperiod, control measurements were done on samples using the samereaction scheme without immobilization of the aminosilane.

One of the theoretical expectations of this study was to see increasingNO-load and release measurements with an increase in branching. However,as demonstrated in FIGS. 19A-19B, NO release measurements weresignificantly higher for SIM-S2 (cumulative release: 43442.53×10⁻¹⁰ molcm-2) when compared to SIM-S4 (cumulative release: 23319.98×10⁻¹⁰ molcm-2) (FIG. 19B, Table 7) over the 25-d period. There could be twopossible explanations for this: steric hindrance in case of higherbranching and hence NAP thiolactone was not able to completely bind tothe amine groups, and/or more branching increased the probability ofchain interactions within the polymer before the free amine groups couldreact with NAP thiolactone. Therefore, it could be concluded thatbranching increasingly doesn't necessarily keep increasing the NO-loador release. This study also demonstrates the design of a surface thatcan release NO up to 25 days at endogenous flux levels. This increasingbranching method is a technique to increase NO release characteristics,much like the function of metal ions when added to NO releasingpolymers. However, beyond the increased NO release properties, thismaterial proved to offer additional advantages as it also impartsantifouling characteristics to the material, as demonstrated by the datadescribed below.

TABLE 5 Contact angle measurements compared between all NAP-thiolactoneand nitroso group functionalized surfaces. Material Static Water ContactAngle (°) SR 106.77 ± 3.36  SIM-N1 94.36 ± 3.36 SIM-S1 101.56 ± 4.48 SIM-N2  64.95 ± 11.87 SIM-S2 53.78 ± 5.23 SIM-N4 93.09 ± 2.91 SIM-S490.90 ± 7.97

TABLE 6 NO Flux release measurements for SIM-S1, SIM-S2, and SIM-S4 upto 600 h. NO Flux (×10⁻¹⁰ mol min⁻¹ cm⁻²) 1 h 4 h 8 h 12 h 36 h 60 h 84h SIM-S1  1.633 ± 0.795 ± 0.566 ± 0.216 ± 0.108 ±  .835 .228 .076 .060.035 SIM-S2  3.733 ± 3.335 ± 3.935 ± 3.677 ± 3.228 ± 3.013 ± 2.391 ± .375 .986 .849 .515 .053 .614 .524 SIM-S4 10.753 ± 4.126 ± 2.946 ±2.547 ± 1.706 ± 1.763 ± 1.122 ± 3.509 .338 .466 .186 .317 .449 .180 NOFlux (×10⁻¹⁰ mol min⁻¹ cm⁻²) 108 h 132 h 156 h 240 h 360 h 480 h 600 hSIM-S1 SIM-S2 1.976 ± 1.493 ± 1.496 ± 1.203 ± .626 ± 0.404 ± 0.305 ±.822 .659 .349 .331 .060 .049 .032 SIM-S4 1.012 ± 0.836 ± 0.673 ± 0.441± 0.316 ± 0.174 ± 0.085 ± .204 .324 .132 .096 .090 .027 .008

TABLE 7 Cumulative NO release measurements for SIM-S1, SIM-S2, andSIM-S4 up to 600 h. Cumulative NO Release (×10⁻¹⁰ mol cm⁻²) 1 h 4 h 8 h12 h 36 h 60 h 84 h 108 h 132 h 156 h 240 h 360 h 480 h 600 h SIM-S1 98316 479 573 807 SIM-S2 223 860 1732 2645 7617 12110 16002 19147 2164523798 30599 37183 40889 43442 SIM-S4 645 1984 2833 3492 6554 9052 1112912666 13997 15084 17894 20621 22386 23320

One common method for assessing the fouling of materials in vitro is toexamine the ability of the material to resist non-specific proteinadhesion. If intended for blood contacting applications, morespecifically, fibrinogen (Fg) adhesion can be assessed. The adsorptionof Fg to the material surface greatly increases the ability foractivated platelets or bacteria to bind to the surface, leading tohigher risks of thrombus formation or infection.₆ While the orientationof Fg adsorption has been shown to determine the degree of plateletadhesion, limiting protein adhesion regardless of orientation isgenerally considered to be an improvement in the hemocompatibility of amaterial.₇ Developing NO-releasing materials that can reduce proteinadsorption could provide drastic improvements in the overallhemocompatibility and antibacterial nature of these materials.₃₀

To examine if the surface immobilized NO-donors (both nitrosated andnon-nitrosated) can provide a decrease in protein adhesion observed onNO-releasing materials, 2 h exposure to FITC-labeled fibrinogen (2 mgmL⁻¹) was conducted at 37° C. (Table 8 and FIG. 20). While minimalchanges in contact angle were observed, increasing the branched natureof the surface grafted NAP groups between SIM-N1 and SIM-N2 decreasedthe degree of Fg adsorption, and is believed to result from increases insteric hindrance.₂₉ However, altering the chemistry of the linkages tothe amine functionalized surface further increases the non-foulingability of these materials. Overall, reductions in protein adsorptionwere observed to reach 65.8±8.9% for SIM-S2 when compared to theunmodified SR. It is also interesting to note that the release of NOfrom the surface had no significant effect on the amount of adsorbed Fg.

Bacterial adhesion, which ultimately results in biofilm formation, is apredominant issue in implanted devices aided by the moist and microbiomesustaining milieu. Coupled with fouling proteins, implants can becomehosts to several pathogens that ultimately lead to medical devicefailure, infection (including bloodstream infection) and sometimesdeath.₈ Antimicrobial efficacy of the designed non-fouling antimicrobialcoating material was compared to the SR control samples (Table 9 andFIG. 21) to confirm their superior bacterial repulsion properties. Thesamples were incubated in bacterial solutions containing ˜10₇-10₈ CFUmL⁻¹ of S. aureus, which is one of the most commonly found nosocomialinfection bacteria._(30, 31) These infections are most commonlyassociated with catheters, stents and prosthetic devices among otherimplants. As mentioned above, it was believed that the NO moleculeswould actively kill bacteria, while the immobilized structure wouldpassively repel proteins and enhance the biocompatibility of thematerial even after all the NO load was exhausted. The antimicrobialefficacy of the designed test samples was clearly observed after 24 h ofincubation, the crucial time for initiation of bacterial infection. TheCFU cm-2 of S. aureus adhered to each material is shown on Table 9.While the non-nitrosated surfaces (SIM-N1 and SIM-N2) demonstrated somereduction in bacterial activity over the control, SIM-S2 showed thehighest bactericidal efficiency with a reduction of 99.91±0.06% (FIG.21) when compared to the SR samples where a growth of ˜10₈ CFU cm⁻² wasobserved. This reduction is higher compared to samples with only NAPthiolactone functionalization (SIM-N1=82.14±22.20% andSIM-N2=96.86±0.49%) and SIM-S1 (85.71±24.74%). It can also be concludedfrom the results that NAP thiolactone functionalized surfaces (SIM-N1and SIM-N2) alone could only reduce bacterial adhesion because theycannot kill bacteria as it does not have any bactericidal property. Thesurfaces functionalized with NO releasing property reduce adhesionthrough passive mechanisms as well as having active antibacterialactivity, thus producing a synergistic effect which enhances theantimicrobial efficacy. In summary, the synergistic effect of themodifiable NO-release kinetics from the SR surface and prevention ofprotein and bacterial adhesion due to the surface immobilized structurescan help significantly reduce undesired clinical consequences of amedical implantation.

Table 9 Shows the Ability of Various Surfaces to Decrease BacterialAdhesion Over 24 h

Platelet activation and adhesion are important considerations whendetermining the hemocompatibility of materials. Upon activation,platelets release several coagulation agonists such as phospholipase A₂(which is then converted into thromboxane A₂), furthering plateletactivation and the coagulation cascade and increasing thrombingeneration.₃₂ One key predecessor of platelet activation is theadsorption of fibrinogen to the materials surface, where changes in theprotein confirmation allows for binding to the platelets Gp IIb/IIIareceptors. Therefore, reducing protein adhesion alone can act as amechanism to reduce platelet activation. Thus, it was demonstrated thatwhile NO releasing materials have been shown to significantly reduceplatelet adhesion, the functionality of the modified surface is not lostafter all the loaded NO has been released. In fact, small molecules withfree thiol groups (similar to NAP) have been shown to provide potentthrombolytic effects when administered systemically by binding to vonWillebrand factor crosslinks of adhered platelets in arterial thrombi.₃₃Both nitrosated and non-nitrosated surfaces were incubated in porcineplatelet rich plasma for 90 min, where platelet adhesion was thendetermined using a lactate dehydrogenase (LDH) assay. The degree ofplatelet adhesion for all materials is shown in FIG. 22. Each variationof the surface modifications was able to provide significant reductionsin platelet adhesion when compared to unmodified SR controls (Table 10).The SIM-N1 and SIM-S2 modifications provided the highest reductions, butwere not statistically significant when compared to each other (pvalue >0.5). However, as the branching increased from the SIM-N1 to theSIM-N2 configuration, the ability to prevent adhesion appeared todecrease (p=0.001). It is believed that this may stem from the chemicalstructure of the methacrylate and diamine linkages. Variation of thecomposition of these to provide a more hydrophilic surface (such asusing an amine-terminated polyethylene-glycol silane, or thepresence/addition of hydroxyl or carboxylic acid side chains) maypossibly allow for further reduction in protein adhesion as the degreeof polymerization at the surface increases. The addition of these sidechains may also provide alternative chemistries for branching. Othernon-limiting options may include diethylenetriamine in place of thepurely alkane backbone of ethylenediamine. While the SIM-S2configuration did not provide significant reductions in plateletadhesion when compared to SIM-N1 or SIM-S1, the significant increase inNO release can provide increased bactericidal activity for extendeddurations.

Table 10: Show the Ability of Various Surfaces to Reduce the PlateletsAdsorbed Per Surface Area Over a Period of 90 Mins

In summary, the present example describes embodiments of the presentdisclosure to attach various amounts of the nitric oxide releasingdonor, SNAP to any polymer material to provide bothbactericidal/antiplatelet activity while simultaneously providing anon-fouling nature to the material surface. This method will be highlyapplicable for biomedical device materials that are prone to infectionsand thrombosis related failures, and can easily be coupled with otherexisting NO-releasing polymers.

It should be emphasized that the above-described embodiments of thepresent disclosure are merely possible examples of implementations, andare set forth only for a clear understanding of the principles of thedisclosure. Many variations and modifications may be made to theabove-described embodiments of the disclosure without departingsubstantially from the spirit and principles of the disclosure. All suchmodifications and variations are intended to be included herein withinthe scope of this disclosure.

REFERENCES FOR EXAMPLE 4

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It should be emphasized that the above-described embodiments of thepresent disclosure are merely possible examples of implementations, andare set forth only for a clear understanding of the principles of thedisclosure. Many variations and modifications may be made to theabove-described embodiments of the disclosure without departingsubstantially from the spirit and principles of the disclosure. All suchmodifications and variations are intended to be included herein withinthe scope of this disclosure.

We claim:
 1. A coated article comprising a nitric oxide-releasingsubstrate having at least one surface; and a zwitterionic polymercovalently attached to the at least one surface to form the coatedarticle; where the zwitterionic polymer is a random copolymer having astructure according to the following formula:

where each occurrence of Z is a zwitterionic moiety; where, in eachinstance, either (i) A¹ is none and A² is ═O or (ii) A¹ is a covalentbond to the at least one surface of the substrate and A² is —OH; whereeach occurrence of R¹ is independently a covalent bond or a linear orbranched, substituted or unsubstituted alkyl diradical having from 1 to12 carbon atoms; where each occurrence of R², R³, and R⁴ isindependently a linear or branched, substituted or unsubstituted alkylhaving from 1 to 12 carbon atoms; and where a, b, and c are real numbersuch that 0<a<1, 0≤b<1, 0<c<1, and a+b+c=1.
 2. The coated articleaccording to claim 1, wherein 0.3≤a≤0.9.
 3. The coated article accordingto claim 1, wherein c is about 0.05.
 4. The coated article according toclaim 1, wherein the nitric oxide-releasing substrate comprises apolymer and nitric-oxide donor dispersed within the polymer; and whereinthe nitric-oxide donor is present in an amount from about 6 wt % toabout 11 wt % based upon a total weight of the nitric oxide-releasingsubstrate.
 5. The coated article according to claim 4, wherein thenitric-oxide donor comprises an organic nitrate, a metal-NO complex, anN-nitrosamine, an S-nitrosothiol, or a combination thereof.
 6. A coatingcomprising: an NO-donor substrate and polymer, wherein the polymerincludes a hydrophilic moiety and a photo cross-linkable moiety, whereinthe polymer is bonded to the NO-donor substrate surface through thephoto cross-linkable moiety, wherein the coating has one or both ofanti-fouling and antimicrobial characteristics, wherein the polymer is azwitterionic polymer that is a random copolymer having a structureaccording to the following formula:

where each occurrence of Z is a zwitterionic moiety; where, in eachinstance, either (i) A¹ is none and A² is ═O or (ii) A¹ is a covalentbond to the at least one surface of the substrate and A² is —OH; whereeach occurrence of R¹ is independently a covalent bond or a linear orbranched, substituted or unsubstituted alkyl diradical having from 1 to12 carbon atoms; where each occurrence of R², R³, and R⁴ isindependently a linear or branched, substituted or unsubstituted alkylhaving from 1 to 12 carbon atoms; and where a, b, and c are real numbersuch that 0<a<1, 0≤b<1, 0<c<1, and a+b+c=1.
 7. The coating of claim 6,wherein the hydrophilic moiety is 2-methacryloyloxyethylphosphorylcholine (MPC).
 8. The coating of claim 6, wherein the polymerincludes an alkyl methacrylate.
 9. The coating of claim 6, wherein thepolymer is selected from one or more of the following:


10. The coating of claim 6, wherein the NO-donor substrate comprises: anorganic nitrate, a metal-NO complex, an N-nitrosamine, a S-nitrosothiol,or a combination thereof.
 11. The coating of claim 6, wherein theNO-donor substrate comprises a polymer doped withS-nitroso-N-acetylpenicillamine.
 12. The coating of claim 11, whereinpolymer doped with S-nitroso-N-acetylpenicillamine includes about 6%-11%wt S-nitroso-N-acetylpenicillamine.
 13. The coating of claim 6, whereinthe polymer forms a film covalently bonded to the NO-donor substratethrough the photo cross-linkable moiety.
 14. The coating of claim 6,wherein the doped polymer film is coated in additional layers of polymerfilm.